Synthesis and use of dual tyrosyl-DNA phosphodiesterase I (Tdp1)—topoisomerase I (Top1) inhibitors

ABSTRACT

The invention described herein pertains to the synthesis and use of certain N-substituted indenoisoquinoline compounds which inhibit the activity Tyrosyl-DNA Phosphodiesterase I (Tdp1) or Topoisomerase I (Top1) or both, or otherwise demonstrate anticancer activity. Also disclosed are novel N-substituted indenoisoquinoline compounds and pharmaceutical compositions comprising the novel N-substituted indenoisoquinoline compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/834,652, filed on Mar. 15, 2013, which claims the benefit ofU.S. Provisional Application No. 61/624,239, filed on Apr. 13, 2012,both of which are incorporated by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under grant numberCA089566 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The invention described herein pertains to the synthesis and use ofcertain N-substituted indenoisoquinoline compounds which inhibit theactivity Tyrosyl-DNA Phosphodiesterase I (Tdp1) or Topoisomerase I(Top1) or both, or otherwise demonstrate anticancer activity.

BACKGROUND AND SUMMARY OF THE INVENTION

As is elaborated below, Topoisomerase I (Top1) and Phosphodiesterase I(Tdp1) are involved in DNA replication, transcription and repair; and itappears that inhibition of either enzyme, or both of them, is useful intreatment of disease, particularly in cancer. Certain N-substitutedindenoisoquinoline compounds have been reported which inhibit theactivity of Top1 and have potential in cancer chemotherapy. See forexample US-2008-0318995-A1.

It has been discovered that N-substituted indenoisoquinoline compoundsare useful as inhibitors of Tdp1 and that some of them inhibit both Tdp1and Top1. Further, novel N-substituted indenoisoquinoline compoundswhich inhibit Top1 and/or show anticancer activity have been discovered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Scheme 1. Tdp1 in Action.

FIG. 2 shows Scheme 2. Tdp1 in Action.

FIG. 3 shows overlapped hypothetical structures of the binary complexTdp1-indenoisoquinoline sulfonate 25 and the crystal structure of thequaternary complex consisting ofTdp1-5′-D(*AP*GP*TP*T)-vanadate-3′-Top1-derived peptide residues 720-727(mutation L724Y). Red: indenoisoquinoline sulfonate; green, Tyr723;yellow, DT806; vanadate, fuchsia. The figure is programmed for wall-eyed(relaxed) viewing.

FIG. 4 shows compounds proposed for synthesis.

FIG. 5 shows Scheme 7. Synthesis of Indenoisoquinoline Sulfonamides.

FIG. 6 shows Scheme 8. Alternative Synthetic Route to IndenoisoquinolineSulfonamides.

FIG. 7 shows Ring-substituted Indenoisoquinoline Sulfonamides. ^(a)Thecytotoxicity GI₅₀ values are the concentrations corresponding to 50%growth inhibition. The MGM is the mean graph midpoint for growthinhibition of all 60 human cancer cell lines successfully tested,ranging from 10⁻⁸ to 10⁻⁴ molar, where values that fall outside therange were taken as 10⁻⁸ and 10⁻⁴ molar.

^(b)Compound-induced DNA cleavage due to Top1 inhibition is graded bythe following semiquantitative relative to 1 μM camptothecin (1): 0, noinhibitory activity; +, between 20 and 50%, activity; ++, between 50 and75% activity; +++, between 75% and 95% activity; ++++, equipotent. The0/+ ranking is between 0 and +.

FIG. 8 shows Scheme 9. Preparation of Bisindenoisoquinolines.

FIG. 9 shows schematic representation of the Tdp1 gel-based assays usingrecombinant Tdp1. The Figure discloses SEQ ID NOS 2 and 3, respectively,in order of appearance.

FIG. 10 shows representative gels showing concentration-dependentinhibition of endogenous Tdp1 in whole cell extract byindenoisoquinoline amine hydrochloride inhibitors. Concentrations were0.5, 1.4, 4.1, 12.3, 37, 111 μM.

FIG. 11 shows inhibitory activities of target compounds against Tdp1 andTop1.

FIG. 12 shows direct binding of 70 to Tdp1 by surface plasmon resonancespectroscopy. Variable concentrations of 70 (33, 11, 3.7, 1.2 and 0.4μM) were injected over amine coupled Tdp1 protein. The compound rapidlyreaches equilibrium and then completely dissociates.

FIG. 13 shows competitive inhibition of 70 against recombinant Tdp1measured by FRET assay.

FIG. 14 shows a titration curve of compound 14 of (B) against Tdp1.

FIG. 15 shows surface plasmon resonance for compound 14 of (B) withTdp1. The binding of compound 14 to Tdp1 was examined using SPRspectroscopy. Different concentrations of 14 (50, 25, 12.5 and 6.25 μM)were injected over immobilized Tdp1. The kinetics were fit to a 1:1binding model yielding the following parameters, k_(a) 7.7 e₄ 1/Ms,k_(d) 0.24 1/s and K_(D) 31 μM.

FIG. 16 shows a hypothetical binding model of 14 of (B) in of thebinding pocket of Tdp1.

FIG. 17 shows a hypothetical binding model of 40 of (B) in of thebinding pocket of Tdp1.

FIG. 18 shows: A. Representative gel showing Tdp1 inhibition forcompounds 54 and 55 of (B). B. Titration curves for determination ofTdp1 IC₅₀ values for compounds 54 and 55 of (B).

FIG. 19 shows Top1-mediated DNA cleavage induced by 85, 77 and 52 of(B). Lane 1: DNA alone; lane 2: Top1 alone; lane 3: 1, 1 μM; lane 4: 5,1 μM; lane 5-16: 85, 77 and 52 at 0.1, 1, 10 and 100 μM respectivelyfrom left to right. Numbers and arrows on the left indicate arbitrarycleavage site positions. The activity of the compounds to produceTop1-mediated DNAcleavage was expressed semiquantitatively as follows:+, weak activity; ++ and +++, moderate activity; ++++, similar activityas 1 μM camptothecin (90).

DETAILED DESCRIPTION

In one embodiment, there is provided a method for treating a disease orcondition responsive to tyrosyl-DNA phosphodiesterase I (Tdp1)inhibition in a host animal, the method comprising the step ofadministering to the host animal an effective amount of a compound ofthe formula

or a pharmaceutically acceptable salt thereof, wherein the compoundreduces the activity of Tdp1, when said compound is in contact withTdp1; and wherein:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12,

R^(T) is amino, methylamino, or dimethylamino, or —(CH₂)_(n)—R^(T)represents

in which:

(N) represents the ring nitrogen;

X is a group having the general structure—(CH₂)_(m)-[(CH₂)_(x)—NR^(M)—(CH₂)_(y)]_(z)—(NR^(N))_(p)—(CH₂)_(q)—,where m is 0 or 1, x and y are integers independently ranging from 1 toabout 4, z is an integer ranging from 1 to about 4, p is 0 or 1, q is 0or an integer ranging from 1 to about 2, and where R^(M) and R^(N) areindependently selected in each instance from hydrogen and methyl;

R^(a) and R^(a′) each independently represent 1-4 substituents each ofwhich is independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted (1-4C)alkyl, optionallysubstituted (1-4C)alkoxy, cyano, nitro, amino, carboxymethylamino,optionally substituted alkylthio, optionally substituted alkylsulfonyl,carboxylic acid and derivatives thereof, and sulfonic acid andderivatives thereof; or R^(a) and R^(a′) each represent 2-4 substituentswhere 2 of said substituents are adjacent substituents and are takentogether with the attached carbons to form an optionally substitutedheterocycle, and where any remaining substituents are each independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy,cyano, nitro, optionally substituted (1-4C)alkylthio, optionallysubstituted (1-4C)alkylsulfonyl, carboxylic acid and derivativesthereof, and sulfonic acid and derivatives thereof; and

R^(d) and R^(d′) each independently represent 1-4 substituents each ofwhich is independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted (1-4C)alkyl, optionallysubstituted (1-4C)alkoxy, cyano, nitro, optionally substituted(1-4C)alkylthio, optionally substituted (1-4C)alkylsulfonyl, phenyl(which may bear one or more amino, hydroxyl, halo, thiol, (1-6C)alkyl orhalo(1-6C)alkyl substituents), carboxylic acid and derivatives thereof,and sulfonic acid and derivatives thereof; or R^(d) and R^(d′) eachindependently represent 2-4 substituents where 2 of said substituentsare adjacent substituents and are taken together with the attachedcarbons to form an optionally substituted heterocycle, and where anyremaining substituents are each independently selected from the groupconsisting of hydrogen, halo, hydroxy, optionally substituted(1-4C)alkyl, optionally substituted (1-4C)alkoxy, cyano, nitro,optionally substituted (1-4C)alkylthio, optionally substituted(1-4C)alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof.

One embodiment of the above method is one wherein R^(a), R^(a′), R^(d),and R^(d′) are independently selected and each includes one or morealkoxy groups or an alkylenedioxy group. One embodiment of the abovemethod is one wherein each of R^(a), R^(a′), R^(d), and R^(d′) ishydrogen.

One embodiment of the above method for any of the above is one wherein Xis CH₂NH(CH₂)₃NHCH₂, CH₂CH₂NH(CH₂)₃NHCH₂CH₂, CH₂CH₂NH(CH₂)₄NHCH₂CH₂, orCH₂NH(CH₂)₂NH(CH₂)₂NHCH₂.

One embodiment of the above method is one wherein the compound is ofFormula A:

or a pharmaceutically acceptable salt thereof, wherein

R is H or (CH₂)_(s)—NH—(CH₂)_(t)—NH—(CH₂)_(u)-A;

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12,

s is 1, 2, or 3;

t is 1, 2, or 3;

u is 1, 2, or 3; and

A is

One embodiment of the above method is one wherein the compound is

wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or apharmaceutically acceptable salt thereof.

One embodiment of the above method is one wherein the compound is

or a pharmaceutically acceptable salt thereof.

One embodiment of the above method is one wherein R^(T) is amino,methylamino, or dimethylamino. Another embodiment for the above methodis one wherein n is 2, 3 or 4. A further embodiment for the above methodis one wherein n is 3.

One embodiment of the above method is one wherein the compound is of theformula

or a pharmaceutically acceptable salt thereof, wherein:R^(T) is amino, R^(a) is 3-amino and R^(d) is hydrogen; orR^(T) is amino, R^(a) is 3-carboxymethylamino and R^(d) is hydrogen; orR^(T) is amino, R^(a) is 3-nitro and R^(d) is 9-methoxy; orR^(T) is amino, R^(a) is 3-amino and R^(d) is 9-methoxy; orR^(T) is amino, R^(a) is 3-nitro and R^(d) is 8-methoxy; orR^(T) is amino, R^(a) is 3-nitro and R^(d) is 7-methoxy; orR^(T) is amino, R^(a) is 3-iodo and R^(d) is 9-methoxy; orR^(T) is dimethylamino, R^(a) is 3-iodo and R^(d) is 9-methoxy; orR^(T) is dimethylamino, R^(a) is 3-cyano and R^(d) is hydrogen.

For any of the above in one embodiment the method is one wherein thedisease or condition responsive to tyrosyl-DNA phosphodiesterase I(Tdp1) inhibition is a cancer.

For any of the above in one embodiment the method is one wherein thehost animal is a human.

In another embodiment of the invention, there is provided a novelcompound of the formula

or a pharmaceutically acceptable salt thereof, wherein:R^(T) is amino, R^(a) is 3-carboxymethylamino and R^(d) is hydrogen; orR^(T) is amino, R^(a) is 3-amino and R^(d) is 9-methoxy; orR^(T) is amino, R^(a) is 3-nitro and R^(d) is 8-methoxy; orR^(T) is amino, R^(a) is 3-nitro and R^(d) is 7-methoxy; orR^(T) is amino, R^(a) is 3-iodo and R^(d) is hydrogen; orR^(T) is dimethylamino, R^(a) is 3-iodo and R^(d) is 9-methoxy; orR^(T) is dimethylamino, R^(a) is 3-cyano and R^(d) is hydrogen.

Another embodiment is a pharmaceutical composition comprising a novelcompound as described above and one or more carriers, diluents, orexcipients, or a combination thereof. A further embodiment is a methodfor treating cancer comprising the step of administering atherapeutically effective amount of a novel compound as described aboveto a patient in need of relief from said cancer.

Another embodiment of the invention is a novel sufonamide compound ofthe formula

or a pharmaceutically acceptable salt thereof, wherein:

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, or 12,

R^(a) represents 1-4 substituents each of which is independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy,cyano, nitro, optionally substituted alkylthio, optionally substitutedalkylsulfonyl, carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(a) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where any remaining substituents are each independently selectedfrom the group consisting of hydrogen, halo, hydroxy, optionallysubstituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy, cyano,nitro, optionally substituted (1-4C)alkylthio, optionally substituted(1-4C)alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof; and

R^(d) represents 1-4 substituents each of which is independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy,cyano, nitro, optionally substituted (1-4C)alkylthio, optionallysubstituted (1-4C)alkylsulfonyl, phenyl (which may bear one or moreamino, hydroxyl, halo, thiol, (1-6C)alkyl or halo(1-6C)alkylsubstituents), carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R^(d) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where any remaining substituents are each independently selectedfrom the group consisting of hydrogen, halo, hydroxy, optionallysubstituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy, cyano,nitro, optionally substituted (1-4C)alkylthio, optionally substituted(1-4C)alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof;

R^(S) is (1-6C)alkyl, (3-7C)cycloalkyl or phenyl, which phenyl may bearone or more amino, hydroxyl, halo, thiol, (1-6C)alkyl or halo(1-6C)alkylsubstituents.

A further embodiment of the above novel sufonamide compound is onewherein:

n is 3;

R^(a) represents 1-2 substituents at the 2-, 3- or 2,3-positions each ofwhich is independently selected from the group consisting of hydrogen,halo, hydroxy, methoxy, cyano and nitro; or R^(a) represents 2substituents where said substituents are adjacent 2,3-substituents andare taken together to form (1-2C)alkylenedioxy; and

R^(d) represents 1-2 substituents at the 8-, 9- or 8,9-positions, eachof which is independently selected from the group consisting ofhydrogen, halo, hydroxy, methoxy and cyano; or R^(d) represents 2substituents where said substituents are adjacent substituents and aretaken together with the attached carbons to form (1-2C)alkylenedioxy;and

R^(S) is methyl or phenyl, which phenyl may bear a 4-bromo or 4-methylsubstituent.

Another embodiment of the above novel sufonamide is the compound of theformula

wherein:

R^(a) represents 3-nitro, 2,3-dimethoxy, or 2,3-methylenedioxysubstituents; R^(d) is hydrogen; and R^(S) is phenyl. In a furtherembodiment R^(a) represents 3-nitro.

Another embodiment of the above novel sufonamide is the compound of theformula

wherein R^(a) is 2-nitro; R^(d) is 9-phenyl; and R^(S) is phenyl.

Another embodiment of the invention is a pharmaceutical compositioncomprising a novel sufonamide compound as described above and one ormore carriers, diluents, or excipients, or a combination thereof. Afurther embodiment of the invention is a method for treating cancercomprising the step of administering a therapeutically effective amountof a novel sufonamide compound as described above to a patient in needof relief from said cancer.

Another embodiment of the invention is a novel compound of the formula

or a pharmaceutically acceptable salt thereof, wherein:R^(T) is dimethylamino, R^(a) is 3-cyano and R^(d) is8,9-methylenedioxy; orR^(T) is 4-morpholinyl, R^(a) is 3-cyano and R^(d) is hydrogen; orR^(T) is imidazolyl, R^(a) is 3-cyano and R^(d) is hydrogen.

Another embodiment of the invention is a pharmaceutical compositioncomprising a novel compound as described above and one or more carriers,diluents, or excipients, or a combination thereof. A further embodimentof the invention is a method for treating cancer comprising the step ofadministering a therapeutically effective amount of a novel compound asdescribed above to a patient in need of relief from said cancer.

In addition, various genera and subgenera of each of R^(a), R_(d),R^(a′), R^(d′), R^(M), R^(N), R^(S), and R^(T), X, and n are describedherein. It is to be understood that all possible combinations of thevarious genera and subgenera of each of R^(a), R^(d), R^(a′), R^(d′),R^(M), R^(N), R^(S), and R^(S), X, and n described herein representadditional illustrative embodiments of compounds of the inventiondescribed herein. It is to be further understood that each of thoseadditional illustrative embodiments of compounds may be used in any ofthe compositions, methods, and/or uses described herein. In anotherembodiment, pharmaceutical compositions containing one or more of thecompounds are also described herein. In one aspect, the compositionsinclude a therapeutically effective amount of the one or more compoundsfor treating a patient with cancer. It is to be understood that thecompositions may include other component and/or ingredients, including,but not limited to, other therapeutically active compounds, and/or oneor more carriers, diluents, excipients, and the like. In anotherembodiment, methods for using the compounds and pharmaceuticalcompositions for treating patients with a cancer are also describedherein. In one aspect, the methods include the step of administering oneor more of the compounds and/or compositions described herein to apatient with a cancer. In another aspect, the methods includeadministering a therapeutically effective amount of the one or morecompounds and/or compositions described herein for treating patientswith a cancer.

It is appreciated herein that the compounds described herein may be usedalone or in combination with other compounds useful for treating acancer, including those compounds that may be therapeutically effectiveby the same or different modes of action.

In each of the foregoing and each of the following embodiments, it is tobe understood that the formulae include and represent not only allpharmaceutically acceptable salts of the compounds, but also include anyand all hydrates and/or solvates of the compound formulae. It isappreciated that certain functional groups, such as the hydroxy, amino,and like groups form complexes and/or coordination compounds with waterand/or various solvents, in the various physical forms of the compounds.Accordingly, the above formulae are to be understood to include andrepresent those various hydrates and/or solvates.

In each of the foregoing and each of the following embodiments, it isalso to be understood that the formulae include and represent eachpossible isomer, such as stereoisomers and geometric isomers, bothindividually and in any and all possible mixtures. In each of theforegoing and each of the following embodiments, it is also to beunderstood that the formulae include and represent any and allcrystalline forms, partially crystalline forms, and non crystallineand/or amorphous forms of the compounds.

The compounds described herein may contain one or more chiral centers,or may otherwise be capable of existing as multiple stereoisomers. It isto be understood that in one embodiment, the invention described hereinis not limited to any particular sterochemical requirement, and that thecompounds, and compositions, methods, uses, and medicaments that includethem may be optically pure, or may be any of a variety of stereoisomericmixtures, including racemic and other mixtures of enantiomers, othermixtures of diastereomers, and the like. It is also to be understoodthat such mixtures of stereoisomers may include a single stereochemicalconfiguration at one or more chiral centers, while including mixtures ofstereochemical configuration at one or more other chiral centers.

Similarly, the compounds described herein may include geometric centers,such as cis, trans, E, and Z double bonds. It is to be understood thatin another embodiment, the invention described herein is not limited toany particular geometric isomer requirement, and that the compounds, andcompositions, methods, uses, and medicaments that include them may bepure, or may be any of a variety of geometric isomer mixtures. It isalso to be understood that such mixtures of geometric isomers mayinclude a single configuration at one or more double bonds, whileincluding mixtures of geometry at one or more other double bonds.

As used herein, halo includes fluoro, chloro, bromo and iodo.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched. It is to be further understood that in certainembodiments, alkyl is advantageously of limited length, including C₁-C₈,C₁-C₆, and C₁-C₄. Illustratively, such particularly limited length alkylgroups, including C₁-C₈, C₁-C₆, and C₁-C₄ may be referred to as loweralkyl. In embodiments of the invention described herein, it is to beunderstood, in each case, that the recitation of alkyl refers to alkylas defined herein, and optionally lower alkyl. Illustrative alkyl,groups are, but not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl,neopentyl, hexyl, heptyl, octyl, and the like.

As used herein, the term “cycloalkyl” includes a chain of carbon atoms,which is optionally branched, where at least a portion of the chain incyclic. It is to be understood that cycloalkylalkyl is a subset ofcycloalkyl. It is to be understood that cycloalkyl may be polycyclic.Illustrative cycloalkyl include, but are not limited to, cyclopropyl,cyclopentyl, cyclohexyl, 2-methylcyclopropyl, cyclopentyleth-2-yl, andthe like.

As used herein, the term “carboxylic acid and derivatives thereof”includes the group CO₂H and salts thereof, and esters and amidesthereof, and CN.

As used herein, the term “sulfonic acid or a derivative thereof”includes SO₃H and salts thereof, and esters and amides thereof.

As used herein, the term “cycloheteroalkyl” including heterocyclyl andheterocycle, includes a chain of atoms that includes both carbon and atleast one heteroatom, such as heteroalkyl, and is optionally branched,where at least a portion of the chain is cyclic. Illustrativeheteroatoms include nitrogen, oxygen, and sulfur. In certain variations,illustrative heteroatoms also include phosphorus, and selenium.Illustrative cycloheteroalkyl include, but are not limited to,tetrahydrofuryl, pyrrolidinyl, tetrahydropyranyl, piperidinyl,morpholinyl, piperazinyl, homopiperazinyl, quinuclidinyl, and the like.

The term “optionally substituted” as used herein includes thereplacement of hydrogen atoms with other functional groups on theradical that is optionally substituted. Such other functional groupsillustratively include, but are not limited to, amino, hydroxyl, halo,thiol, alkyl, haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl,heteroaryl, heteroarylalkyl, heteroarylheteroalkyl, nitro, sulfonicacids and derivatives thereof, carboxylic acids and derivatives thereof,and the like. Illustratively, any of amino, hydroxyl, thiol, alkyl,haloalkyl, heteroalkyl, aryl, arylalkyl, arylheteroalkyl, heteroaryl,heteroarylalkyl, heteroarylheteroalkyl, and/or sulfonic acid isoptionally substituted.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. It is to beunderstood that the compositions described herein may be prepared fromisolated compounds described herein or from salts, solutions, hydrates,solvates, and other forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from variousamorphous, non-amorphous, partially crystalline, crystalline, and/orother morphological forms of the compounds described herein. It is alsoto be understood that the compositions may be prepared from varioushydrates and/or solvates of the compounds described herein. Accordingly,such pharmaceutical compositions that recite compounds described hereinare to be understood to include each of, or any combination of, thevarious morphological forms and/or solvate or hydrate forms of thecompounds described herein. Illustratively, compositions may include oneor more carriers, diluents, and/or excipients. The compounds describedherein, or compositions containing them, may be formulated in atherapeutically effective amount in any conventional dosage formsappropriate for the methods described herein. The compounds describedherein, or compositions containing them, including such formulations,may be administered by a wide variety of conventional routes for themethods described herein, and in a wide variety of dosage formats,utilizing known procedures (see generally, Remington: The Science andPractice of Pharmacy, (21^(st) ed., 2005)).

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known to the researcher, veterinarian, medical doctoror other clinician of ordinary skill.

It is also appreciated that the therapeutically effective amount,whether referring to monotherapy or combination therapy, isadvantageously selected with reference to any toxicity, or otherundesirable side effect, that might occur during administration of oneor more of the compounds described herein. Further, it is appreciatedthat the co-therapies described herein may allow for the administrationof lower doses of compounds that show such toxicity, or otherundesirable side effect, where those lower doses are below thresholds oftoxicity or lower in the therapeutic window than would otherwise beadministered in the absence of a cotherapy.

In addition to the illustrative dosages and dosing protocols describedherein, it is to be understood that an effective amount of any one or amixture of the compounds described herein can be readily determined bythe attending diagnostician or physician by the use of known techniquesand/or by observing results obtained under analogous circumstances. Indetermining the effective amount or dose, a number of factors areconsidered by the attending diagnostician or physician, including, butnot limited to the species of mammal, including human, its size, age,and general health, the specific disease or disorder involved, thedegree of or involvement or the severity of the disease or disorder, theresponse of the individual patient, the particular compoundadministered, the mode of administration, the bioavailabilitycharacteristics of the preparation administered, the dose regimenselected, the use of concomitant medication, and other relevantcircumstances.

The dosage of each compound of the claimed combinations depends onseveral factors, including: the administration method, the condition tobe treated, the severity of the condition, whether the condition is tobe treated or prevented, and the age, weight, and health of the personto be treated. Additionally, pharmacogenomic (the effect of genotype onthe pharmacokinetic, pharmacodynamic or efficacy profile of atherapeutic) information about a particular patient may affect thedosage used.

It is to be understood that in the methods described herein, theindividual components of a co-administration, or combination can beadministered by any suitable means, contemporaneously, simultaneously,sequentially, separately or in a single pharmaceutical formulation.Where the co-administered compounds or compositions are administered inseparate dosage forms, the number of dosages administered per day foreach compound may be the same or different. The compounds orcompositions may be administered via the same or different routes ofadministration. The compounds or compositions may be administeredaccording to simultaneous or alternating regimens, at the same ordifferent times during the course of the therapy, concurrently individed or single forms.

The term “administering” as used herein includes all means ofintroducing the compounds and compositions described herein to thepatient, including, but are not limited to, oral (po), intravenous (iv),intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal,ocular, sublingual, vaginal, rectal, and the like. The compounds andcompositions described herein may be administered in unit dosage formsand/or formulations containing conventional nontoxicpharmaceutically-acceptable carriers, adjuvants, and/or vehicles.

Illustrative formats for oral administration include tablets, capsules,elixirs, syrups, and the like.

Illustrative routes for parenteral administration include intravenous,intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal,intramuscular and subcutaneous, as well as any other art recognizedroute of parenteral administration.

Illustrative means of parenteral administration include needle(including microneedle) injectors, needle-free injectors and infusiontechniques, as well as any other means of parenteral administrationrecognized in the art. Parenteral formulations are typically aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably at a pH in the range from about 3 to about9), but, for some applications, they may be more suitably formulated asa sterile non-aqueous solution or as a dried form to be used inconjunction with a suitable vehicle such as sterile, pyrogen-free water.The preparation of parenteral formulations under sterile conditions, forexample, by lyophilization, may readily be accomplished using standardpharmaceutical techniques well known to those skilled in the art.Parenteral administration of a compound is illustratively performed inthe form of saline solutions or with the compound incorporated intoliposomes. In cases where the compound in itself is not sufficientlysoluble to be dissolved, a solubilizer such as ethanol can be applied.

When given systemically, such as parenterally, illustrative dosesinclude those in the range from about 0.01 mg/kg to about 100 mg/kg, orabout 0.01 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 100mg/kg, or about 0.1 mg/kg to about 10 mg/kg.

When given systemically, such as orally, illustrative doses includethose in the range from about 0.1 mg/kg to about 1000 mg/kg, or about0.1 mg/kg to about 100 mg/kg, or about 0.1 mg/kg to about 10 mg/kg, orabout 1 mg/kg to about 1000 mg/kg, or about 1 mg/kg to about 100 mg/kg,or about 1 mg/kg to about 10 mg/kg.

In another illustrative embodiment, the compound is administeredparenterally systemically q.d. at a dose of about 0.1 mg/kg, or about0.5 mg/kg, or about 1 mg/kg, or about 5 mg/kg, or about 10 mg/kg, orabout 50 mg/kg of body weight of the patient.

In making the pharmaceutical compositions of the compounds describedherein, a therapeutically effective amount of one or more compounds inany of the various forms described herein may be mixed with one or moreexcipients, diluted by one or more excipients, or enclosed within such acarrier which can be in the form of a capsule, sachet, paper, or othercontainer. Excipients may serve as a diluent, and can be solid,semi-solid, or liquid materials, which act as a vehicle, carrier ormedium for the active ingredient. Thus, the formulation compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders. The compositions may contain anywhere from about 0.1% to about99.9% active ingredients, depending upon the selected dose and dosageform.

Illustrative examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,alginates, tragacanth, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxybenzoates; sweetening agents; and flavoring agents. Thecompositions can be formulated so as to provide quick, sustained ordelayed release of the active ingredient after administration to thepatient by employing procedures known in the art. It is to be understoodthat one or more carriers, one or more diluents, one or more excipients,and combinations of the foregoing may be used in making thepharmaceutical compositions described herein. It is appreciated that thecarriers, diluents, and excipients used to prepare the compositionsdescribed herein are advantageously GRAS (generally regarded as safe)compounds.

Illustrative examples of emulsifying agents include naturally occurringgums (e.g., gum acacia or gum tragacanth) and naturally occurringphosphatides (e.g., soybean lecithin and sorbitan monooleatederivatives). Examples of antioxidants are butylated hydroxy anisole(BHA), ascorbic acid and derivatives thereof, tocopherol and derivativesthereof, butylated hydroxy anisole, and cysteine. Examples ofpreservatives are parabens, such as methyl or propyl p-hydroxybenzoate,and benzalkonium chloride. Examples of humectants are glycerin,propylene glycol, sorbitol, and urea. Examples of penetration enhancersare propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide,N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof,tetrahydrofurfuryl alcohol, and AZONE. Examples of chelating agents aresodium EDTA, citric acid, and phosphoric acid. Examples of gel formingagents are CARBOPOL, cellulose derivatives, bentonite, alginates,gelatin and polyvinylpyrrolidone. Examples of ointment bases arebeeswax, paraffin, cetyl palmitate, vegetable oils, sorbitan esters offatty acids (Span), polyethylene glycols, and condensation productsbetween sorbitan esters of fatty acids and ethylene oxide (e.g.,polyoxyethylene sorbitan monooleate (TWEEN)).

Solid Dosage Forms for Oral Use. Formulations for oral use includetablets containing the active ingredient(s) in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose, sorbitol, sugar,mannitol, microcrystalline cellulose, starches including potato starch,calcium carbonate, sodium chloride, lactose, calcium phosphate, calciumsulfate, or sodium phosphate); granulating and disintegrating agents(e.g., cellulose derivatives including microcrystalline cellulose,starches including potato starch, croscarmellose sodium, alginates, oralginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia,alginic acid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). Furthermore, a time delaymaterial such as, e.g., glyceryl monostearate or glyceryl distearate maybe employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology.

Controlled Release Oral Dosage Forms. Controlled release compositionsfor oral use may, e.g., be constructed to release the active drug bycontrolling the dissolution and/or the diffusion of the active drugsubstance.

Parenteral Compositions. The pharmaceutical composition may also beadministered parenterally by injection, infusion or implantation(intravenous, intramuscular, subcutaneous, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

As indicated above, the pharmaceutical compositions described herein maybe in the form suitable for sterile injection. To prepare such acomposition, the suitable active drug(s) are dissolved or suspended in aparenterally acceptable liquid vehicle. Among acceptable vehicles andsolvents that may be employed are water, water adjusted to a suitable pHby addition of an appropriate amount of hydrochloric acid, sodiumhydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, andisotonic sodium chloride solution. The aqueous formulation may alsocontain one or more preservatives (e.g., methyl, ethyl or n-propylp-hydroxybenzoate). In cases where one of the compounds is onlysparingly or slightly soluble in water, a dissolution enhancing orsolubilizing agent can be added, or the solvent may include 10-60% w/wof propylene glycol or the like.

The examples in the following disclosures further illustrate specificembodiments of the invention; however, the following illustrativeexamples should not be interpreted in any way to limit the invention.

Further aspects and embodiments of the invention are set out in thefollowing descriptions, published at (A) J. Med. Chem. 2012, 55,4457-4478 and (B) J. Med. Chem. 2013, 56, 182-200, each of which isincorporated by reference.

Abbreviations used herein include the following: APCI-MS,atmospheric-pressure chemical ionization mass spectrometry; BBO,multinuclear broadband observe; CI/EIMS, chemical ionization/electronimpact mass spectrometry; CPT, camptothecin; DMAP,4-dimethylaminopyridine; DMSO-d₆, dimethyl-d₆ sulfoxide; EIMS, electronimpact mass spectrometry; ESIMS or ESI-MS, electrospray ionization massspectrometry; HRMS, high resolution mass spectrometry; PTSA,p-toluenesulfonic acid; QNP, quattro nucleus probe; SCAN1,spinocerebellar ataxia with axonal neuropathy; Tdp1, tyrosyl-DNAphosphodiesterase I; TFA, trifluoroacetic acid; Top1, topoisomerase typeI; TsCl, p-toluenesulfonyl chloride.

(A) J. Med. Chem. 2012, 55, 4457-4478

Eukaryotic topoisomerase I (Top1) is an essential enzyme for manycritical cellular processes as it relaxes the double helix structure ofDNA so that the stored genetic information can be accessed during DNAreplication, transcription and repair. The mechanism of action of Top1starts with the nucleophilic attack of the enzyme Tyr723 hydroxyl groupon a phosphodiester linkage in DNA, displacing the 5′-end to becomecovalently attached to the 3′-end of DNA, thus forming a “cleavagecomplex.” The religation reaction occurs faster than cleavage so theequilibrium favors the uncleaved DNA (FIG. 1, Scheme 1).

Under normal circumstances, the Top1-DNA cleavage complex is atransitory intermediate in the Top1-catalyzed reaction, as the brokenDNA strand is quickly religated after a local supercoil has beenremoved. However, Top1 can become stalled in the DNA cleavage complexunder a variety of natural or unnatural conditions in which the rate ofreligation is inhibited or reduced. For example, Top1 inhibitors, suchas camptothecin (CPT, 1) and its clinically used derivatives (topotecan(2), irinotecan (3), and belotecan), as well as other non-CPT Top1inhibitors like indenoisoquinolines (indotecan (4), and indimitecan(5)), inhibit the

religation rate by selectively and reversibly binding to the Top1-DNAinterface. This ultimately leads to cell death after collision of thecleavage complex with the replication fork resulting in double-strandbreakage. Other naturally occurring DNA lesions, such as strand breaks,abasic sites, base mismatches, and certain oxidized or modified bases,can also induce stalled Top1-DNA complexes via the misalignment of the5′-hydroxyl with the tyrosyl-DNA phosphodiester linkage, thus physicallyblocking the Top1 religation reaction. Under these conditions, cellularDNA metabolism results in repair of the stalled Top1-DNA cleavagecomplex by DNA ligase, which cannot work until the protein adduct isremoved, and the broken DNA strand is provided with termini consistingof a 5′-phosphate on one end and a 3′-hydroxyl on the other end for DNArepair. In detail, the overall process involves the following steps:1) Tdp1 hydrolyzes the phosphotyrosyl linkage between degraded Top1 andDNA;2) polynucleotide kinase phosphatase (PNKP) hydrolyzes the resulting3′-phosphate end and catalyzes the phosphorylation of the 5′-hydroxylend of the broken DNA strand. This results in a broken DNA strand withtermini consisting of a 5′-phosphate and 3′-hydroxyl for DNA repair.3) DNA polymerase β replaces the missing DNA segment; and finally 4) DNAligase III reseals the broken DNA.

Tyrosyl-DNA phosphodiesterase I (Tdp1) has been shown to be the onlyenzyme that specifically catalyzes the hydrolysis of the phosphodiesterbond between the catalytic Tyr723 of Top1 and DNA-3′-phosphate. Hence,Tdp1 is thought to be associated with the repair of DNA lesions. Thecellular importance of Tdp1 also stems from the fact it is ubiquitous ineukaryotes and plays an important physiological role, as the homozygousmutation H493R in its active site is responsible for the rare autosomalrecessive neurodegenerative disease called spinocerebellar ataxia withaxonal neuropathy (SCAN1). Tdp1 also has the ability to remove the3′-phosphoglycolate caused by oxidative DNA damage and bleomycin andrepair trapped Top2-DNA cleavage complexes. All this evidence suggeststhat Tdp1 assumes a broader role in the maintenance of genomicstability. Hence, this makes Tdp1 a rational anticancer drug developmenttarget.

Tdp1 is a member of the phospholipase D superfamily of enzymes thatcatalyze the hydrolysis of a variety of phosphodiester bonds in manydifferent substrates. Crystallographic studies have revealed that humanTdp1 is composed of two domains related by a pseudo-twofold axis ofsymmetry. Each domain contributes a histidine and a lysine residue toform an active site that is centrally located at the symmetry axis. Fouradditional residues N283, Q294, N516, and E538 are also positioned nearthe active site. The crystal structure of Tdp1 in the quaternary complexwith a vanadate ion, a Top1-derived peptide, and a single-stranded DNAoligonucleotide revealed an active site in which the DNA moiety occupiesa relatively narrow cleft rich in positive charges, while the peptidemoiety binds in another region of the active site characterized by arelatively large, more open cleft that contains a mixed chargedistribution. The trigonal bipyramidal geometry exhibited by thevanadate implies an S_(N)2 mechanism for nucleophilic attack onphosphate. Therefore, the mechanism of action of Tdp1 is proposed tostart with a nucleophilic attack on the phosphotyrosyl bond by thecatalytic H263 residue in the N-terminal domain while the H493 residuein the C-terminal domain acts as a general acid and donates a proton tothe tyrosine-containing peptide leaving group (FIG. 2, Scheme 2). Theresulting phosphoramide is stabilized by hydrogen-bonding with catalyticK265 and K495. Hydrolysis of this covalent intermediate occurs via asecond S_(N)2 reaction by a water molecule with the H493 residue actingas a general base. This proposed reaction step is supported by in vitrostudies showing that the SCAN1 H493R mutation leads to an accumulationof Tdp1-DNA covalent intermediate. The final product in this process isa DNA molecule with a 3′-phosphate end.

Because the Top1-DNA phosphotyrosyl bond is buried deep within theTop1-DNA complex and is inaccessible to Tdp1, prior denaturation of theTop1-DNA complex or proteolytic degradation of Top1 is required for theTdp1 enzymatic activity. However, Tdp1 seems to be equally effectiveagainst many structural variations of DNA, including single-strand,tailed duplex, and gapped duplex, though the activity decreases as theoligonucleotide length is shortened. These observations have impliedthat the enzymatic activity of Tdp1 is influenced by the length ofTop1-derived polypeptide chain and the structure of the DNA segmentbound to Top1. Moreover, studies from SCAN1 cells provided evidence forTdp1 participation in the repair of Top1-mediated DNA damage and for thehypersensitivity to camptothecin in human cells with a single defect inTdp1 activity. These observations suggest the possibility of developingTdp1 inhibitors that can potentiate the cytotoxic effects of Top1inhibitors in anticancer drug therapy.

To date, there are very few known Tdp1 inhibitors, and their potenciesand specificities leave much room for improvement. For example, bothvanadate and tungstate can mimic the phosphate in the transition state,thus expressing inhibition at millimolar concentrations. However, due topoor specificity and hypersensitivity to all phosphoryl transferreactions, they cannot serve as pharmacological inhibitors. Other Tdp1inhibitors are the aminoglycoside neomycin, which has very low potencyat IC₅₀=8 mM, or furamidine (6), which produces reversible andcompetitive inhibition of Tdp1 with an IC₅₀≈30 μM. However, 6 hasadditional targets because of its DNA binding activities, which alsomakes experimental data difficult to interpret. The steroid NSC 88915(7) was recently identified via high-throughput screening as a potentand specific Tdp1 inhibitor with an IC₅₀=7.7 μM.

However, this compound expressed some common pharmacokinetic problems incellular systems such as limited drug uptake, poor cytotoxicity, andoff-target effects. Therefore, there is an urgent need to design anddevelop potent Tdp1 inhibitors that can overcome these drawbacks.Furthermore, potential anticancer agents that would possess both Tdp1and Top1 inhibitory activities are attractive because the two types ofactivities could act synergistically.

Screening of a focused library of indenoisoquinolines led to thediscovery of three potent Tdp1 inhibitors (Table 1). These Tdp1-activen-alkylamino-containing indenoisoquinolines 77-79 (n=10-12), which hadbeen synthesized previously by Morrell et al., did not display Top1inhibition or were very weak inhibitors, although other homologouscompounds with shorter linkers (69-71, n=2-4) were good Top1 inhibitors.This led to the idea that Tdp1 inhibition may potentially be present inTop1 inhibitors with a shorter side chain.

TABLE 1 Tdp1 Inhibitory Activities of n-Alkylamino IndenoisoquinolinesCompound 77-79

IC₅₀ (μM) n recombinant Tdp₁ ^(a)Top₁ 10 14 0 11 13 + 19 15 0/+^(a)Compound-induced DNA cleavage due to Top1 inhibition is graded bythe following semiquantitative relative to 1 μM camptothecin (1): 0, noinhibitory activity; +, between 20 and 50%, activity; ++, between 50 and75% activity; +++, between 75% and 95% activity; ++++, equipotent. The0/+ ranking is between 0 and +.In addition, previous studies reported the importance of the sulfonatefunctional group in conferring the Tdp1 inhibitory activity of steroid 7since this group mimics the phosphotyrosyl bond in the Top1-DNA complex.This led to the hypothesis that sulfonate analogues of theindenoisoquinolines in Table 1 might function as Tdp1 inhibitors. Thishypothesis was also supported by GOLD docking and energy minimization ofone hypothetical sulfonate (25, n=3) in the crystal structure of Tdp1(1RFF) after deleting the polydeoxyribonucleotide 5′-D(*AP*GP*TP*T)-3′,vanadate (VO₄ ³⁻), and the Top1-derived peptide residues 720-727(mutation L724Y). The docking results revealed a structure thatresembles the original structure: the tosylate moiety matched thephosphotyrosine the sulfonate was in place of the vanadate, the alkylchain played the “spacer” role of the deoxyribose, and theindenoisoquinoline system overlapped with a thymine of DNA (FIG. 3).This result is in agreement with those reported for steroid 7. Since asulfonamide bond is well known to be more metabolically stable thansulfonate, and the current compounds possess a structural handle (aminogroup) that can be used to install a sulfonyl linkage, a series ofindenoisoquinoline sulfonates and sulfonamides with different linkerlengths (n=2-12) were synthesized in order to investigate how thestructures of these indenoisoquinolines resemble the substrate of theTdp1-catalyzed reaction. Moreover, a previous study on steroid 7indicated that different substituents on the phenyl ring may havepositive effects on Tdp1 inhibitory activity. Therefore, four differentgroups were placed at the sulfonamide position (FIG. 4).

Synthesis of Indenoisoquinoline Sulfonates

The key starting material in the synthesis of all of the desiredcompounds is lactone 11, which was synthesized based on the procedurereported by Morrell et al. The reaction of 2-carboxybenzaldehyde (8) andphthalide (9) in the presence of sodium methoxide in methanol yieldedthe intermediate 10, which formed lactone 11 upon cyclization underacidic conditions in a one-pot synthesis carried out with the aid of aDean-Stark trap. The synthetic route to indenoisoquinoline sulfonates24-29 involved the preparation of n-hydroxyalkyl indenoisoquinolines18-23 from the reaction of lactone 11 and n-aminoalcohols 12-17 for 3-6hours. The sulfonylation of compounds 19-23 to afford indenoisoquinolinesulfonates 25-29 was achieved in dichloromethane (Scheme 3).

However, the sulfonylation of 2-hydroxyethyl indenoisoquinoline 18 underthe same conditions failed to yield the desired sulfonate product 24,but instead gave the chloride analogue 30 or, in the presence of silveracetate, the acetate analogue 31 (Scheme 4). Silver acetate wasinitially meant to quench the nucleophilic attack of the chloride ionsin the hope of obtaining the sulfonate 24, and the side product 31 wasunexpected.

Two mechanisms are proposed for this transformation with the sulfonate24 assumed to be the first intermediate (Scheme 5). In the firstproposed mechanism, 24 undergoes intramolecular cyclization andelimination of the tosylate group to yield a 4,5-dihydro-3-oxazoliumintermediate 32. Nucleophilic attack of the chloride anion yields thesubstituted 2-chloroethyl indenoisoquinoline 30. The presence of silverion facilitates the displacement of chloride by acetate to form 31. Inthe second mechanism, chloride displaces the tosylate directly to form31. This mechanism does not explain why the alcohol 18 undergoes aunique reaction pathway.

An independent study of the feasibility of displacing the chloride byacetate was done by mixing and stirring chloride 30 and silver acetatein dichloromethane without the presence of any base at room temperaturefor 24 hours. The result showed that 30 was incompletely converted tothe acetate 31 and, surprisingly, to alcohol 18. This result implied thesensitivity of acetate 31 to hydrolysis and its instability in solution.

7-Hydroxyheptyl indenoisoquinoline 23 could be prepared from lactone 11and the commercially available 7-amino-1-heptanol (17) as depicted inScheme 3. However, due to the very high cost of 17 (and anyn-aminoalcohol with more than 6 methylene units) several alternativeroutes were considered for making 23. Aminoalcohol 17 can be readilyprepared from the reaction of its low-cost 7-bromo analogue 33 withsodium azide in CHCl₃ to afford an azido intermediate, which yields 17upon reduction with Fe in aqueous ammonia (Scheme 6A). However, sincethis route poses an explosion hazard because of the instability of theazido compound and the possible formation of the very explosive di- andtriazidomethane (from CHCl₃) during work-up, an alternative route wasutilized to make 17 under safer and milder conditions using theclassical Delépine reaction (Scheme 6B). In this method, urotropine 34was alkylated by the 7-bromoalcohol 33 to produce a quaternaryhexamethylenetetramine salt 35, which produced the 7-aminoalcohol 17 ingood yield upon acidic hydrolysis in ethanol. Another advantage of thismethod is that intermediate 17 requires no purification and can reactwith lactone 11 to afford 23 in very high purity.

Synthesis of Indenoisoquinoline Sulfonamides

All the desired n-alkylamino indenoisoquinoline hydrochloride salts69-79 were synthesized based on the procedures reported by Morrell etal. as follows: the diamines 36-46 were Boc protected at one end byusing a 5:1 diamine:Boc₂O ratio. The mono-Boc-protected products 47-57were treated with lactone 11 in a 1:1 ratio to give the Boc-protectedindenoisoquinolines 58-68 in high yields, which, upon deprotection ofthe amines in methanolic HCl, provided the indenoisoquinolinehydrochloride salts 69-79. All of the indenoisoquinoline sulfonamideswith methyl (80-90), phenyl (91-101), p-methylphenyl (102-112), andp-bromophenyl (113-123) substituents were synthesized in good toexcellent yields by gently heating each hydrochloride salt at 70° C.with a corresponding sulfonating reagent (mesyl, benzenesulfonyl, tosyl,or p-bromobenzenesulfonyl chloride) in 1:2 ratio in the presence oftriethylamine (2 equiv) for 16 h (FIG. 5, Scheme 7).

A different synthetic route to the indenoisoquinoline sulfonamides wasinvestigated as follows: 1) the sulfonamide group was incorporated intothe linker chain by having the sulfonating reagents (mesyl,benzenesulfonyl, tosyl, or p-bromobenzenesulfonyl chloride) react withdiamines in a 1:5 molar ratio, with the procedure for these reactionsbeing similar to that of making mono-Boc-protected diamines; 2) heatingthe mono-sulfonated diamines with lactone 11 in a 1:1 molar ratio underreflux to obtain the desired sulfonamides (FIG. 6, Scheme 8). Theadvantage of this approach is that the introduction and removal of theprotecting Boc group were skipped, thus providing a shorter route tomake sulfonamides with hypothetically higher overall yields. However,the apparent weakness is that derivatization of side chains at thesulfonamide end would be impossible. Nevertheless, this idea was firsttested by the reaction of N-(3-aminopropyl)-4-methylbenzenesulfonamide(124) with lactone 11 to give sulfonamide 103 in 70% yield. Followingthis success, N-(11-aminoundecyl)-4-methylbenzenesulfonamide (125) andN-(11-aminoundecyl)benzenesulfonamide (126) were synthesized to use inthe next reaction with lactone 11. However, the condensation occurredvery slowly in CHCl₃ and a significant amount of starting materials wasobserved even after 48 h of heating at reflux. Then benzene was employedto help remove H₂O from the reaction mixture in order to force theequilibrium to the product side, but the reaction provided only about30% yield of crude product after 2 days of heating, and startingmaterials were still present in the mixture. Hence, the original methodwas utilized to make all of the desired sulfonamides 80-123.

Synthesis of Bisindenoisoquinolines and Other Compounds

The goal of this project was to achieve dual Top1-Tdp1 inhibition, andthis was attempted by making sulfonamides from substrates that have beenshown to be active against Top1 by previous biological assays (FIG. 7).The presence of a bulky substituent at position 9 on the D-ring of theindenoisoquinoline system attenuates Top1 inhibition, as seen incompounds 127 and 129 (see structure 127 in FIG. 7 forindenoisoquinoline numbering). Nevertheless, in order to gain a quicklook into the effects of various substituents on the A- and D-rings ofthe indenoisoquinoline system to Tdp1 inhibitory activity, all thestarting materials 127-131 were subjected to similar reaction conditionsto obtain the corresponding sulfonamides 132-136 (FIG. 7). In addition,three bis(indenoisoquinlines) 140-142 were prepared by heating twoequivalents of lactone 11 with diamines 137-139 at reflux for 16 h.Polyamino bis(indenoisoquinline) 145 was synthesized based on theprocedure reported by Morrell et al. (FIG. 8, Scheme 9).

Biological Results

Tdp1 inhibitory activity was measured by the drug's ability to inhibitthe hydrolysis of the phosphodiester linkage between tyrosine and the3′-end of the DNA substrate, and to prevent the generation of anoligonucleotide with a free 3′-phosphate (N14P). Therefore, thedisappearance of the gel band for N14P indicated Tdp1 inhibition. TheTdp1 catalytic gel-based assay is represented in FIG. 9, andrepresentative gels demonstrating dose-dependent Tdp1 inhibition by someindenoisoquinoline amine hydrochlodrides are depicted in FIG. 10.

Additionally, all indenoisoquinoline sulfonates and sulfonamideas weretested for Top1 cleavage complex poisoning in a Top1-mediated DNAcleavage assay. The potency of a compound against Top1 is correlated tothe intensities of the bands corresponding to DNA fragments in thisassay, and is graded by the following semiquantitative scale relative to1 μM camptothecin: 0, no inhibitory activity; +, between 20% and 50%activity; ++, between 50% and 75% activity; +++, between 75% and 95%activity; ++++, equipotent. The Tdp1 and Top1 activities of all targetcompounds are represented in FIG. 11.

The indenoisoquinoline amine hydrochlorides 69-79 were shown to be Tdp1inhibitors (IC₅₀=13 to 55 μM). Despite being less active than steroid 7(IC₅₀=7.7 μM), their potencies were retained in whole cell extract(WCE), while the steroid expressed off-target effects and lost itspotency in cellular environments. Notably, compounds with longer linkers(n=10-12) showed higher Tdp1 inhibition than those with shorter sidechains, while the Top1 inhibition trend was the opposite. It is knownthat the optimal length of the side chain at the lactam position in Top1indenoisoquinoline inhibitors is three methylene units (propyl).Molecular modeling indicated that an increase in hydrophobicity as adirect result of increased linker length in these Top1 inhibitors causednegative interactions with the hydrated binding pocket of the Top1-DNAcleavage complex. That explained the decrease in Top1 inhibition of thisseries as the number of methylene units increase. This is, however, notthe case for Tdp1 inhibition. Though the present results for Tdp1inhibition do not show a linear correlation of chain length and potency,they indicate that the longer chains may gain favorable hydrophobicinteractions in the binding site of Tdp1. Additionally, hypotheticalmodels show that the lactam side chain protrudes towards the majorgroove of DNA and causes minimal steric clashes within the bindingpocket in the Top1-DNA cleavage complex. Similar conclusions can bedrawn for Tdp1: since no significant reduction in activity was observedin the series with increasing chain length, the lactam side chain mustbe well accommodated. Indenoisoquinolines with n=3 and 4 (compounds 70and 71) with IC₅₀=22 to 29 μM, MGM GI₅₀=0.16 to 0.32 μM, Top1 inhibition“+++” are the most potent representatives of the 69-79 series tested fordual Top1-Tdp1 inhibitory activity. These also represent a new chemotypeof fully synthetic small-molecule Tdp1 inhibitors.

Surprisingly, the hydroxyl analogues (compound 18-23) were inactiveagainst Tdp1. Despite being similar in ability to form hydrogen bonds,the discrepancy in Tdp1 potency when going from the amino group to thehydroxyl group in this series lends support to the hypothesis thathydrogen bonding may not be a predominant factor that determines Tdp1inhibitory activity. Moreover, it seems reasonable to consider theseamino inhibitors to be protonated at physiological pH, and thispositively charged state, which is not possible in the hydroxyl series18-23, seems to be an important requirement for Tdp1 inhibition. Thisrationale is in agreement with a previous report that the terminal aminogroup in this series is important for cellular cytotoxicity, though notabsolutely necessary for Top1 inhibition.

One member of this amino series (70, n=3) was subjected to SurfacePlasmon Resonance (SPR) assays based on reported procedures in order togain a more detailed understanding of the Tdp1-inhibitor interaction andto measure the affinity of 70 to Tdp1 (FIG. 12). The binding of theinhibitor to the surface-bound Tdp1 induces an increase in resonanceunits from the initial baseline until a steady-state phase of thisinteraction is achieved as depicted by a plateau. The decrease inresonance units corresponds to the dissociation or reversibility of theinteraction. This result demonstrates direct binding of 70 to Tdp1. Thiscompound was also evaluated using a Fluorescence Resonance EnergyTransfer (FRET) assay and was found to be a competitive inhibitor withK_(i)=3.19 μM (FIG. 13). Steroid 7, which binds Tdp1 directly, is alsocompetitive with the DNA substrate for the Tdp1 active site.

All indenoisoquinoline sulfonates and sulfonamides (compounds 80-123)were inactive against Tdp1 (IC₅₀>111 μM). This result was unexpectedsince extensive studies on steroid 7 clearly emphasized the importanceof the sulfonyl ester moiety for Tdp1 inhibition, and our initialmodeling (FIG. 3) also suggested a good mimic of the phosphotyrosinelinkage by the sulfonate group, which is similar to how steroid 7resembles this group. As the sulfonate or sulfonamide moiety wasattached to the terminal amino group, the activity was completelyabolished independently of the linker length (n=2-12). Similarly,sulfonamides 132-136 were also inactive against Tdp1. Although stericclashes between the sulfonate or sulfonamide group with components ofthe binding pocket might be responsible for this result, thisexplanation is not satisfactory because even when a short side chain ispresent such as in compound 80, which bears a relatively small mesylategroup on an ethyl linker, the activity drops drastically. In the case ofthe amino series (compounds 69-79), the ligand-binding site seems toaccommodate a long side chain quite well. Therefore, steric clashescould only explain the inactivity of compounds with very long and bulkyside chains. A different factor must have been the cause of thesignificant attenuation of Tdp1 inhibition upon sulfonylation of theamino series even though this was completely different in case ofsteroid 7. Due to the lack of a crystal structure of Tdp1 in complexwith an inhibitor, no firm conclusion can be drawn as to where a ligandbinds and how it inhibits Tdp1 activity, and molecular modeling failedto provide an answer to the question of sulfonate and sulfonamideinactivities in the present series.

Among the four bis(indenoisoquinolines) synthesized and tested, only thepolyamino bis(indenoisoquinoline) 145 displayed low micromolar Tdp1inhibition, with an IC₅₀=1.5 μM and 1.9 μM against rec. and WCE Tdp1,respectively. Three other bis(indenoisoquinolines) 140-142, which weremade because of the initial positive results from the amino series with10-12 methylene linkers, were inactive. This result provides additionalsupport to the hypothesis that the protonation of the amino groups maygive favorable charge-complementary interactions within the Tdp1 bindingpocket, thereby conferring Tdp1 inhibition. The polyaminobis(indenoisoquinoline) 145 is currently the most potent dual Top1-Tdp1inhibitor, displaying Top1 inhibition (“++++”) equipotent tocampothecin, excellent antiproliferative potency (MGM GI₅₀=0.394 μM),and also excellent inhibitory activity against Tdp1 in both human rec.and WCE Tdp1 with IC₅₀=1.5 and 1.9 μM, respectively.

A series of bis(indenoisoquinolines) and indenoisoquinolines with amino,sulfonate, and sulfonamide side chains have been synthesized to evaluatethe hypothesis that dual Top1-Tdp1 inhibition can be achieved in asingle compound. In contrast with the reported importance of thesulfonyl ester moiety to the Tdp1 inhibition, all sulfonates andsulfonamides were inactive, while the free amines at various linkerlengths displayed good to excellent inhibition. Among them, twocompounds 70 and 71 were potent against both Tdp1 and Top1, representingthe first two dual Top1-Tdp1 inhibitors ever reported. Significantinsights for future lead optimization were deduced: 1) hypotheticalcharge-complementary interactions between protonated amino groups withinthe Tdp1 active site may contribute to high potency, and 2) thehydrophobicity of the polymethylene moiety linking the amino group tothe heterocycle may also contribute to activity. The polyaminobis(indenoisoquinoline) 145 is currently the most potent dual Top1-Tdp1inhibitor. This encouraging result has much significance because: 1)this class of indenoisoquinoline compounds serves as the first evidencethat having Top1 and Tdp1 inhibitory activity in one single smallmolecule is in fact possible; 2) the unique structural features ofindenoisoquinolines allow much room for manipulation so thepharmacokinetics (absorption, distribution, and excretion) can bemodulated and optimized in ways that are not possible for other types ofTdp1 inhibitors, 3) they represent lead molecules for development of newdual Top1-Tdp1 inhibitory agents, and 4) they provide a set ofinhibitory ligands that could possibly be crystallized in complex withTdp1, which would facilitate the structure-based drug design approach.

Experimental Section

General.

Solvents and reagents were purchased from commercial vendors and wereused without any further purification. Melting points were determinedusing capillary tubes with a Mel-Temp apparatus and were uncorrected.Infrared spectra were obtained using KBr pellets using CHCl₃ as thesolvent. IR spectra were recorded using a Perkin-Elmer 1600 series orSpectrum One FTIR spectrometer. ¹H NMR spectra were recorded at 300 MHzusing a Bruker ARX300 spectrometer with a QNP probe. Mass spectralanalyses were performed at the Purdue University Campus-Wide MassSpectrometry Center. ESI-MS studies were performed using a FinniganMATLCQ Classic mass spectrometer. EI/CI-MS studies were performed using aHewlett-Packard Engine or GCQ FinniganMAT mass spectrometer. APCI-MSstudies were carried out using an Agilent 6320 Ion Trap massspectrometer. Combustion microanalyses were performed at the PurdueUniversity Microanalysis Laboratory using a Perkin-Elmer Series IICHNS/O model 2400 analyzer. All reported values are within 0.4% of thecalculated values. Analytical thin layer chromatography was carried outon Baker-flex silica gel IB2-F plates, and compounds were visualizedwith short wavelength UV light and ninhydrin staining. Silica gel flashchromatography was performed using 230-400 mesh silica gel. HPLCanalyses were performed on a Waters 1525 binary HPLC pump/Waters 2487dual λ absorbance detector system using a 5 μM C₁₈ reverse phase column.Purities of biologically important compounds were ≧95%. For puritiesestimated by HPLC, the major peak accounted for ≧95% of the combinedtotal peak area when monitored by a UV detector at 254 nm. All yieldsrefer to isolated compounds.

Benz[d]indeno[1,2-b]pyran-5,11-dione (11)

Sodium metal (3.678 g, 0.160 mol) was cut into small pieces and added toMeOH (40 mL) to make a 4 M methanolic solution, which was then added toa solution of 2-carboxybenzaldehyde (8) (1.000 g, 6.661 mmol) andphthalide (9) (0.893 g, 6.661 mmol) in ethyl acetate (20 mL). Themixture was stirred and heated at 70° C. for 16 h to yield an orangesolution, which was then concentrated, diluted with H₂O (100 mL),acidified with 10% HCl until pH 1, and extracted with EtOAc (50 mL×3).The organic layers were combined and extracted with 1 N NaOH (50 mL×3).The aqueous layers were combined and acidified with concentrated HCluntil pH 1 to give a red solution. The acidic mixture was extracted withethyl acetate (50 mL×3) and washed with brine (50 mL). The organiclayers were dried over anhydrous MgSO₄, filtered, and concentrated toyield the intermediate 10. The crude intermediate 10 was dissolved inbenzene (125 mL), followed by an addition of TsOH.H₂O (100 mg). Theresulting mixture was heated for 7 h at reflux in a flask affixed with aDean-Stark trap. The solution was cooled to room temperature,concentrated, diluted with CHCl₃ (150 mL), and washed with sat NaHCO₃(50 mL×3) and brine (50 mL). The organic layer was dried over anhydrousNa₂SO₄, and concentrated to yield the desired product as an orange solid(1.69 g, 93%): mp 254-256° C. (lit. 257° C.). ¹H NMR (300 MHz, CDCl₃) δ8.39 (d, J=7.8 Hz, 1H), 8.31 (d, J=8.2 Hz, 1H), 7.84 (t, J=7.5 Hz, 1H),7.61 (d, J=6.9 Hz, 1H), 7.55-7.39 (m, 4H).

General Procedure for the Preparation of n-HydroxyalkylIndenoisoquinolines 18-22

n-Aminoalcohols 12-16 (0.50 g) in CHCl₃ (10 mL) were added to a solutionof lactone 11 (1.0 equiv) in CHCl₃ (50 mL). The reaction mixtures wereheated at reflux for 3-6 h with stirring, and then washed with H₂O (50mL×2) and brine (50 mL). The organic layers were dried over anhydrousNa₂SO₄, filtered and concentrated, adsorbed onto SiO₂, and purified byflash column chromatography (SiO₂), eluting with 5% MeOH in CHCl₃, toprovide the products 18-22 in high purity.

6-(2-Hydroxyethyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (18)

The general procedure provided the desired product as a red solid (1.10g, 62%): mp 201-204° C. (lit. 200-201° C.). ¹H NMR (300 MHz, CDCl₃) δ8.68 (d, J=8.1 Hz, 1H), 8.32 (d, J=8.3 Hz, 1H), 7.74 (dt, J=1.2 and 7.1Hz, 1H), 7.62 (dd, J=1.2 and 7.0 Hz, 2H), 7.48-7.37 (m, 3H), 4.76 (t,J=5.8 Hz, 2H), 4.21 (q, J=5.6 Hz, 2H), 2.60 (m, J=5.3 Hz, 1H); ESI-MSm/z (rel intensity) 292 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 292.0974.found: 292.0978; HPLC purity: 96.6% (MeOH, 100%), 96.2% (MeOH—H₂O,90:10).

6-(3-Hydroxypropyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (19)

The general procedure provided the desired product as a red solid (1.20g, 98%): mp 173-175° C. (lit. 170-171° C.). ₁H NMR (300 MHz, CDCl₃) δ8.75 (d, J=8.2 Hz, 1H), 8.38 (d, J=8.1 Hz, 1H), 7.79-7.65 (m, 3H),7.53-7.41 (m, 3H), 4.76 (t, J=6.4 Hz, 2H), 3.75 (q, J=6.0 Hz, 2H), 3.26(t, J=6.3 Hz, 1H), 2.20 (m, 2H); ESI-MS m/z (rel intensity) 328 (MNa⁺,61); HRMS (+ESI) calcd for MH⁺: 306.1130. found: 306.1127; HPLC purity:99.5% (MeOH, 100%), 98.6% (MeOH—H₂O, 90:10).

6-(4-Hydroxybutyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (20)

The general procedure provided the desired product as a red solid (1.23g, 95%): mp 165-166° C. (lit. 160-162° C.). ¹H NMR (300 MHz, CDCl₃) δ8.73 (d, J=8.1 Hz, 1H), 8.36 (d, J=8.3 Hz, 1H), 7.76 (dt, J=1.2 and 8.2Hz, 1H), 7.65 (dd, J=1.4 and 6.7 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H),7.50-7.38 (m, 3H), 4.62 (t, J=7.7 Hz, 2H), 3.84 (q, J=6.0 Hz, 2H), 2.07(m, 2H), 1.86 (m, 2H), 1.70 (t, J=5.3 Hz, 1H); ESI-MS m/z (relintensity) 320 (MH⁺, 42); HRMS (+ESI) calcd for MH⁺: 320.1287. found:320.1289; HPLC purity: 99.1% (MeOH, 100%), 96.2% (MeOH—H₂O, 90:10).

6-(5-Hydroxypentyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)dione (21)

The general procedure provided the desired product as an orange solid(1.45 g, 90%): mp 144-146° C. (lit. 146-148° C.). ¹H NMR (300 MHz,CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.35 (d, J=8.4 Hz, 1H), 7.75 (dt, J=1.2and 7.0 Hz, 1H), 7.65 (dd, J=1.4 and 4.9 Hz, 1H), 7.49-7.38 (m, 4H),4.56 (t, J=7.7 Hz, 2H), 3.74 (q, J=5.3 Hz, 2H), 1.98 (m, 2H), 1.74-1.60(m, 4H), 1.42 (t, J=4.8 Hz, 1H); ESI-MS m/z (rel intensity) 334 (MH⁺,100); HRMS (+ESI) calcd for MH⁺: 334.1443. found: 334.1445; HPLC purity:97.9% (MeOH, 100%), 95.4% (MeOH—H₂O, 90:10).

6-(6-Hydroxyhexyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (22)

The general procedure provided the desired product as a red solid (1.24g, 84%): mp 139-141° C. IR (film) 3425, 1765, 1698, 1663, 1611, 1550,1504, 1427, 1317, 758 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz,1H), 8.36 (d, J=8.2 Hz, 1H), 7.75 (dt, J=1.3 and 7.4 Hz, 1H), 7.65 (dd,J=1.0 and 7.2 Hz, 1H), 7.50-7.37 (m, 4H), 4.56 (t, J=7.7 Hz, 2H), 3.70(q, J=5.7 Hz, 2H), 3.50 (d, J=4.9 Hz, 1H), 1.96 (m, 2H), 1.66-1.45 (m,6H); ESI-MS m/z (rel intensity) 370 (MNa⁺, 100); HRMS (+ESI) calcd forMNa⁺: 370.1419. found: 370.1424; HPLC purity: 97.6% (MeOH, 100%), 99.0%(MeOH—H₂O, 90:10).

6-(7-Hydroxyheptyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (23)

Bromoalcohol 33 (0.975 g, 5.0 mmol) and urotropine (34, 0.771 g, 5.5mmol) dissolved in CHCl₃ (20 mL). The mixture was stirred at reflux for5 h, and allowed to stand overnight to induce the formation ofquaternary salt 35. The salt was filtered and added to a 2 M ethanolicHCl solution (15 mL). The reaction mixture was warmed up gently with aheat gun, and swirled to produce white NH₄Cl precipitate, which wasremoved by filtration. The mother liquor was concentrated in vacuo anddiluted in H₂O (20 mL), followed by cooling in an ice bath. The solutionwas made strongly alkaline (pH 13) with 6 M NaOH, extracted with diethylether (25 mL×3), and washed with brine (25 mL). The ethereal layers werecombined, dried over anhydrous Na₂SO₄, and concentrated to afford ayellowish oil of crude 17. The crude 17 was dissolved in CHCl₃ (20 mL)and added to a solution of lactone 11 (1 equiv of 33) in CHCl₃ (50 mL).The reaction mixture was heated at reflux for 18 h, concentrated,adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂),eluting with EtOAc-hexane in a gradient of concentration ratios from 5:3to 7:3 to provide the desired product as a fine red powdery solid (0.95g, 52%): mp 132-135° C. IR (film) 3468, 1776, 1698, 1663, 1611, 1550,1504, 1427, 1318, 756 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz,1H), 8.36 (d, J=8.1 Hz, 1H), 7.72 (t, J=7.0 Hz, 1H), 7.65 (d, J=6.4 Hz,1H), 7.49-7.40 (m, 4H), 4.54 (t, J=7.8 Hz, 2H), 3.67 (d, J=3.4H, 2H),1.94-1.86 (m, 2H), 1.62-1.43 (m, 8H), 1.30 (m, 1H); ESI-MS m/z (relintensity) 362 (MH⁺, 17); HRMS (+ESI) calcd for MH⁺: 362.1756. found:362.1760. Anal. calcd for C₂₃H₂₃NO₃.0.2H₂O: C, 75.68; H, 6.46; N, 3.84.Found: C, 75.51; H, 6.32; N, 3.62.

General Procedure for the Preparation of Indenoisoquinoline Sulfonates25-29 and Chloride 30

A solution of Et₃N (2 equiv) in CH₂Cl₂ (1 mL) and DMAP (0.2 equiv) wasadded to solutions of the n-alkylhydroxy indenoisoquinolines 19-23 (100mg) in CH₂Cl₂ (10 mL). The solutions were stirred at room temperaturefor 5 min, and tosyl chloride (2 equiv) was added. The reaction mixtureswere stirred at room temperature for 16 h, quenched with aq 3 M HCl (50mL), and washed with H₂O (50 mL), sat. NaHCO₃ (50 mL) and brine (50 mL).The organic layers were dried over anhydrous Na₂SO₄, filtered andconcentrated, purified by flash column chromatography (SiO₂), elutingwith EtOAc (3-5%) in CHCl₃, to provide the indenoisoquinoline sulfonates25-29 in high purity after trituration with diethyl ether (20 mL).

3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl-4-methyl-benzenesulfonate(25)

The general procedure provided the desired product as a red solid (132.5mg, 88%): mp 177-179° C. (lit. 180-182° C.). ¹H NMR (300 MHz, CDCl₃) δ8.72 (d, J=8.1 Hz, 1H), 8.31 (d, J=7.5 Hz, 1H), 7.83 (d, J=8.3 Hz, 2H),7.73-7.63 (m, 3H), 7.49-7.34 (m, 5H), 4.62 (t, J=7.9 Hz, 2H), 4.31 (t,J=5.7 Hz, 2H), 2.46 (s, 3H), 2.34 (m, 2H); ESI-MS m/z (rel intensity)482 (MNa⁺, 100); HRMS (+ESI) calcd for MNa⁺: 482.1038. found: 482.1041;HPLC purity: 98.6% (MeOH, 100%), 99.4% (MeOH—H₂O, 90:10).

4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)butyl-4-methyl-benzenesulfonate(26)

The general procedure provided the desired product as an orange solid(129 mg, 87%): mp 160-163° C. IR (film) 1757, 1695, 1667, 1612, 1550,1504, 1428, 1348, 1174, 753 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d,J=8.0 Hz, 1H), 8.32 (d, J=7.4 Hz, 1H), 7.78-7.70 (m, 3H), 7.66 (d, J=7.0Hz, 1H), 7.50-7.42 (m, 4H), 7.33 (d, J=8.2 Hz, 2H), 4.55 (t, J=6.8 Hz,2H), 4.16 (t, J=5.9 Hz, 2H), 2.42 (s, 3H), 1.98-1.88 (m, 4H); ESIMS m/z(rel intensity) 496 (MNa⁺, 100); HRMS (+ESI) calcd for MNa⁺: 496.1195.found: 496.1201; HPLC purity: 98.4% (MeOH, 100%), 99.2% (MeOH—H₂O,90:10).

5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)pentyl-4-methyl-benzenesulfonate(27)

The general procedure provided the desired product as an orange solid(119 mg, 81%): mp 163-165° C. IR (film) 1697, 1661, 1609, 1549, 1503,1427, 1355, 1174, 755 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz,1H), 8.34 (d, J=7.8 Hz, 1H), 7.79-7.70 (m, 3H), 7.66 (d, J=6.8 Hz, 1H),7.50-7.39 (m, 4H), 7.34 (d, J=8.1 Hz, 2H), 4.51 (t, J=7.8 Hz, 2H), 4.09(t, J=6.1 Hz, 2H), 2.43 (s, 3H), 1.91-1.75 (m, 4H), 1.65-1.57 (m, 2H);ESI-MS m/z (rel intensity) 488 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺:488.1532. found: 488.1539; HPLC purity: 97.1% (MeOH, 100%), 98.0%(MeOH—H₂O, 90:10).

6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)hexyl-4-methyl-benzenesulfonate(28)

The general procedure provided the desired product as an orange solid(123 mg, 85%): mp 146-149° C. IR (film) 1767, 1693, 1662, 1610, 1550,1503, 1428, 1357, 1176, 757 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d,J=8.2 Hz, 1H), 8.34 (d, J=7.9 Hz, 1H), 7.80-7.70 (m, 3H), 7.65 (d, J=6.8Hz, 1H), 7.49-7.38 (m, 4H), 7.36 (d, J=8.0 Hz, 2H), 4.51 (t, J=7.6 Hz,2H), 4.07 (t, J=6.3 Hz, 2H), 2.44 (s, 3H), 1.88 (m, 2H), 1.73 (m, 2H),1.57 (m, 4H); ESI-MS m/z (rel intensity) 502 (MH⁺, 100); HRMS (+ESI)calcd for MH⁺: 502.1688. found: 502.1694; HPLC purity: 98.0% (MeOH,100%), 96.8% (MeOH—H₂O, 90:10).

7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)heptyl-4-methyl-benzenesulfonate(29)

The general procedure provided the desired product as a red solid (109mg, 76%): mp 123-126° C. IR (film) 1775, 1698, 1664, 1611, 1550, 1504,1428, 1358, 1176, 758 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz,1H), 8.35 (d, J=7.6 Hz, 1H), 7.80-7.70 (m, 3H), 7.65 (d, J=6.8 Hz, 1H),7.49-7.39 (m, 4H), 7.35 (d, J=8.1 Hz, 2H), 4.51 (t, J=7.7 Hz, 2H), 4.05(t, J=6.3 Hz, 2H), 2.44 (s, 3H), 1.90 (m, 2H), 1.67 (m, 2H), 1.49 (m,2H), 1.40-1.37 (m, 4H); ESI-MS m/z (rel intensity) 516 (MH⁺, 100); HRMS(+ESI) calcd for MH⁺: 516.1845. found: 516.1848; HPLC purity: 95.4%(MeOH, 100%), 98.3% (MeOH—H₂O, 90:10).

6-(2-Chloroethyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (30)

The general procedure provided the chloride product as a purple solid(105 mg, 99%): mp 210-212° C. (lit. 197-199° C.). ¹H NMR (300 MHz,CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.2 Hz, 1H), 7.78 (dt, J=1.2and 8.1 Hz, 1H), 7.67 (d, J=6.8 Hz, 1H), 7.59 (d, J=7.4 Hz, 1H),7.52-7.42 (m, 3H), 4.86 (t, J=7.4 Hz, 2H), 3.97 (t, J=7.7 Hz, 2H);probe-EI/CI-MS m/z (rel intensity) 309 (M⁺, 33); HRMS (+EI/CI) calcd forM⁺: 309.0557. found: 309.0560; HPLC purity: 95.3% (MeOH, 100%), 97.3%(MeOH—H₂O, 90:10).

2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin6(11H)-yl)ethyl Acetate (31)

Et₃N (34.7 mg, 0.343 mmol), CH₃COOAg (57.2 mg, 0.343 mmol), and TsCl(65.4 mg, 0.343 mmol) were added to the solution of 18 (50 mg, 0.172mmol) in CH₂Cl₂ (50 mL). The reaction mixture was stirred at roomtemperature for 16 h, and then washed with sat. NaHCO₃ (50 mL) and brine(50 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated, adsorbed onto SiO₂, and purified by flash columnchromatography (SiO₂), eluting with EtOAc—CHCl₃ (2:8) to afford thedesired product as a red solid (38.5 mg, 67%): mp 221-224° C. IR (film)1738, 1693, 1655, and 1506 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.75 (d,J=8.0 Hz, 1H), 8.37 (d, J=7.9 Hz, 1H), 7.75 (t, J=5.8 Hz, 2H), 7.67 (dd,J=1.1 and 5.8 Hz, 1H), 7.51-7.41 (m, 3H), 4.84 (t, J=6.2 Hz, 2H), 4.57(t, J=6.1 Hz, 2H), 1.94 (s, 3H); probe-EI/CI-MS m/z (rel intensity) 334(MH⁺, 100); HRMS (+EI/CI) calcd for M⁺: 333.1001. found: 333.1005; HPLCpurity: 97.3% (MeOH, 100%), 98.0% (MeOH—H₂O, 90:10).

Compounds 47-79 were synthesized based on the procedures reported byMorrell at al. Purities of biologically tested indenoisoquinoline aminehydrochlorides 69-79 were ≧95% by HPLC.

TABLE 2 Purities of Indenoisoquinoline Amine Hydrochlorides 69-79 byHPLC Purity by HPLC Comp. MeOH, 100% MeOH—H₂O, 90:10 69 97.8 97.6 7096.7 98.0 71 98.5 100 72 98.3 100 73 98.1 97.8 74 95.7 100 75 96.9 98.576 95.5 95.8 77 99.4 98.8 78 98.7 96.6 79 99.2 98.5

General Procedure for the Preparation of Indenoisoquinoline Sulfonamides80-123

Indenoisoquinoline salts 69-79 (50 mg, 0.107-0.153 mmol) were dissolvedin CHCl₃ (15 mL), followed by the addition of Et₃N (2 equiv) in CHCl₃ (1mL). The solutions were stirred at room temperature for 5 min. Mesyl,benzenesulfonyl, tosyl, or p-bromobenzenesulfonyl chloride (2 equiv)were then added. The mixtures were heated at reflux for 16 h, and thenwashed with sat. NaHCO₃ (50 mL) and brine (50 mL). The organic layerswere dried over anhydrous Na₂SO₄, filtered and concentrated, andpurified by flash column chromatography (SiO₂), eluting with EtOAc—CHCl₃to provide the indenoisoquinoline sulfonamide products in high purity.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-methanesulfonamide(80)

The crude product was eluted with EtOAc—CHCl₃ (4:6) to afford thedesired product as an orange solid (38.7 mg, 69%): mp 250-255° C. IR(film) 3313, 1705, 1653, 1609, 1549, 1502, 1418, 1321, 1129, 758 cm⁻¹;¹H NMR (300 MHz, DMSO-d₆) δ 8.59 (d, J=8.0 Hz, 1H), 8.23 (d, J=8.0 Hz,1H), 7.89-7.82 (m, 2H), 7.58-7.47 (m, 5H), 4.61 (t, J=6.7 Hz, 2H), 3.42(m, 2H), 2.91 (s, 3H); probe-EI/CI-MS m/z (rel intensity) 369 (M⁺, 100);HRMS (EI/CI) calcd for M⁺: 368.0831. found: 368.0835; HPLC purity: 96.0%(MeOH, 100%), 96.3% (MeOH—H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)propyl)-methanesulfonamide(81)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (47.1 mg, 84%): mp 201-203° C. IR(film) 3203, 1696, 1646, 1610, 1549, 1504, 1428, 1317, 1137, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.32 (d, J=8.1 Hz,1H), 7.77 (t, J=7.0 Hz, 1H), 7.65 (d, J=6.5 Hz, 1H), 7.55-7.38 (m, 4H),5.73 (t, J=6.8 Hz, 1H), 4.70 (t, J=6.0 Hz, 2H), 3.25 (q, J=6.5 Hz, 2H),3.00 (s, 3H), 2.23-2.23 (m, 2H); APCI-MS m/z (rel intensity) 383 (MH⁺,100); HRMS (+ESI) calcd for MH⁺: 383.1066. found: 383.1069; HPLC purity:99.1% (MeOH, 100%), 96.7% (MeOH—H₂O, 90:10).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)butyl)-methanesulfonamide(82)

The crude product was eluted with EtOAc—CHCl₃ (7:3) to afford thedesired product as an orange solid (42.2 mg, 81%): mp 193-194° C. IR(film) 3272, 2346, 1694, 1655, 1611, 1549, 1504, 1318, 1152, 756 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=7.8 Hz, 1H), 8.34 (d, J=8.3 Hz,1H), 7.74 (t, J=6.8 Hz, 1H), 7.66 (d, J=6.9 Hz, 1H), 7.50-7.40 (m, 4H),4.60 (m, 3H), 3.34 (q, J=6.6 Hz, 2H), 3.00 (s, 3H), 2.05 (m, 2H), 1.85(m, 2H); ESI-MS m/z (rel intensity) 397 (MH⁺, 100); HRMS (+ESI) calcdfor MH⁺: 397.1222. found: 397.1231; HPLC purity: 98.4% (MeOH, 100%),97.2% (MeOH—H₂O, 90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)pentyl)-methanesulfonamide(83)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (44.0 mg, 79%): mp 150-152° C. IR(film) 3275, 1698, 1660, 1611, 1550, 1504, 1318, 1150, 756 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz, 1H), 8.34 (d, J=8.0 Hz, 1H), 7.76(td, J=7.1 and 1.1 Hz, 1H), 7.65 (d, J=6.9 Hz, 1H), 7.50-7.41 (m, 4H),4.57 (t, J=7.4 Hz, 2H), 4.48 (t, J=5.9 Hz, 1H), 3.24 (q, J=6.4 Hz, 2H),2.97 (s, 3H), 2.00 (m, 2H), 1.76 (m, 2H), 1.65 (m, 2H); ESI-MS m/z (relintensity) 411 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 411.1379. found:411.1381; HPLC purity: 99.7% (MeOH, 100%), 98.5% (MeOH—H₂O, 90:10).

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)hexyl)-methanesulfonamide(84)

The crude product was eluted with EtOAc—CHCl₃ (5:5) to afford thedesired product as an orange solid (42.7 mg, 77%): mp 160-161° C. IR(film) 3355, 1697, 1646, 1608, 1548, 1504, 1452, 1364, 1258, 764 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.2 Hz, 1H), 8.36 (d, J=7.6 Hz,1H), 7.76 (dd, J=7.0 and 1.2 Hz, 1H), 7.65 (d, J=6.8 Hz, 1H), 7.50-7.41(m, 4H), 4.56 (t, J=7.6 Hz, 1H), 4.44 (m, 1H), 3.20 (q, J=6.5 Hz, 2H),2.97 (s, 3H), 1.93 (m, 2H), 1.67 (m, 2H), 1.54 (m, 4H); ESI-MS m/z (relintensity) 425 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 425.1538. found:425.1532; HPLC purity: 99.8% (MeOH, 100%), 98.3% (MeOH—H₂O, 90:10).

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)heptyl)-methanesulfonamide(85)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (33 mg, 60%): mp 159-162° C. IR(film) 3227, 1687, 1646, 1609, 1547, 1505, 1429, 1308, 1144, 754 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.36 (d, J=8.2 Hz,1H), 7.73 (dd, J=1.3 and 6.9 Hz, 1H), 7.65 (d, J=6.7 Hz, 1H), 7.50-7.39(m, 4H), 4.54 (t, J=7.8 Hz, 2H), 4.24 (m, 1H), 3.19 (q, J=6.8 Hz, 2H),2.97 (s, 3H), 1.91 (m, 2H), 1.63-1.43 (m, 8H); ESI-MS m/z (relintensity) 461 (MNa⁺, 100); HRMS (+ESI) calcd for MNa⁺: 461.1511. found:461.1518. Anal. calcd for C₂₄H₂₆N₂O₄S.0.75H₂O: C, 63.77; H, 6.13; N,6.20. Found: C, 63.73; H, 6.06; N, 5.86.

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)octyl)-methanesulfonamide(86)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (41.3 mg, 75%): mp 151-155° C. IR(film) 3214, 1767, 1699, 1645, 1612, 1551, 1504, 1426, 1315, 1142, 760cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.0Hz, 1H), 7.75 (t, J=7.1 Hz, 1H), 7.66 (d, J=6.8 Hz, 1H), 7.50-7.39 (m,4H), 4.54 (t, J=8.2 Hz, 2H), 4.24 (m, 1H), 3.17 (q, J=6.9 Hz, 2H), 2.96(s, 3H), 1.90 (m, 2H), 1.57-1.26 (m, 10H); ESI-MS m/z (rel intensity)453 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 453.1848. found: 453.1853.Anal. calcd for C₂₅H₂₈N₂O₄S: C, 66.35; H, 6.24; N, 6.19. Found: C,66.34; H, 6.31; N, 5.81.

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)nonyl)-methanesulfonamide(87)

The crude product was eluted with EtOAc—CHCl₃ (5:5) to afford thedesired product as an orange solid (54.2 mg, 82%): mp 154-156° C. IR(film) 3211, 1700, 1645, 1550, 1506, 1426, 1314, 1142, 759 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.36 (d, J=7.7 Hz, 1H), 7.73(t, J=8.2 Hz, 1H), 7.66 (d, J=6.2 Hz, 1H), 7.50-7.40 (m, 4H), 4.54 (t,J=8.0 Hz, 2H), 4.21 (m, 1H), 3.17 (q, J=6.9 Hz, 2H), 2.96 (s, 3H), 1.90(m, 2H), 1.63-1.23 (m, 12H); ESI-MS m/z (rel intensity) 467 (MH⁺, 50);HRMS (+ESI) calcd for MH⁺: 467.2005. found: 467.2007. Anal. calcd forC₂₆H₃₀N₂O₄S.0.2H₂O: C, 66.41; H, 6.52; N, 5.96. Found: C, 66.18; H,6.39; N, 5.70.

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)decyl)-methanesulfonamide(88)

The crude product was eluted with EtOAc—CHCl₃ (3:7) to afford thedesired product as an orange solid (45.3 mg, 83%): mp 115-117° C. IR(film) 3297, 1697, 1663, 1611, 1551, 1505, 1428, 1351, 1175, 759 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.35 (d, J=7.5 Hz,1H), 7.72 (t, J=6.9 Hz, 1H), 7.65 (d, J=6.3 Hz, 1H), 7.49-7.40 (m, 4H),4.53 (t, J=7.9 Hz, 2H), 4.20 (br s, 1H), 3.16 (q, J=6.8 Hz, 2H), 2.95(s, 3H), 1.93 (m, 2H), 1.56-1.18 (m, 14H); ESI-MS m/z (rel intensity)481 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 481.2161. found: 481.2165;HPLC purity: 95.7% (MeOH, 100%), 96.2% (MeOH—H₂O, 90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)undecyl)-methanesulfonamide(89)

The crude product was eluted with EtOAc—CHCl₃ (5:5) to afford thedesired product as an orange solid (48.7 mg, 89%): mp 136-137° C. IR(film) 3299, 1699, 1658, 1612, 1577, 1504, 1424, 1312, 1134, 761 cm⁻¹;¹H-NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.2 Hz, 1H), 8.35 (d, J=8.1 Hz,1H), 7.75 (t, J=7.3 Hz, 1H), 7.65 (d, J=6.3 Hz, 1H), 7.49-7.40 (m, 4H),4.53 (t, J=7.8 Hz, 2H), 4.25 (br s, 1H), 3.16 (q, J=6.6 Hz, 2H), 2.96(s, 3H), 1.90 (m, 2H), 1.61-1.25 (m, 16H); ESI-MS m/z (rel intensity)495 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 495.2318. found: 495.2322;HPLC purity: 97.4% (MeOH, 100%), 100% (MeOH—H₂O, 90:10).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)dodecyl)-methanesulfonamide(90)

The crude product was eluted with EtOAc—CHCl₃ (5:5) to afford thedesired product as an orange solid (58.0 mg, 89%): mp 113-117° C. IR(film) 3268, 1698, 1662, 1610, 1550, 1504, 1427, 1318, 1151, 759 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.71 (d, J=8.0 Hz, 1H), 8.35 (d, J=8.0 Hz,1H), 7.74 (t, J=7.7 Hz, 1H), 7.64 (d, J=6.5 Hz, 1H), 7.49-7.40 (m, 4H),4.52 (t, J=7.7 Hz, 2H), 4.26 (m, 1H), 3.16 (dd, J=13 and 6.7 Hz, 2H),2.95 (s, 3H), 1.89 (m, 14H); APCI-MS m/z (rel intensity) 509 (MH⁺, 100);HRMS (+ESI) calcd for MNa⁺: 531.2294. found: 531.2291; HPLC purity:99.2% (CH₃CN, 100%), 98.3% (CH₃CN—H₂O, 90:10).

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-benzenesulfonamide(91)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (47.8 mg, 72%): mp 246-249° C. IR(film) 3233, 1702, 1652, 1610, 1551, 1505, 1425, 1342, 1157, 756 cm⁻¹;¹H NMR (300 MHz, DMSO-d₆) δ 8.54 (d, J=8.1 Hz, 1H), 8.18-8.13 (m, 2H),7.80-7.72 (m, 4H), 7.58-7.48 (m, 7H), 4.55 (t, J=6.9 Hz, 2H), 3.23 (t,J=6.8 Hz, 2H); ESI-MS m/z (rel intensity) 431 (MH⁺, 13); HRMS (+ESI)calcd for MH⁺: 431.1066. found: 431.1073; HPLC purity: 95.9% (MeOH,100%), 98.0% (MeOH—H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)-benzenesulfonamide(92)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as a red solid (57.2 mg, 88%): mp 216-218° C. IR (film)3282, 1655, 1611, 1550, 1502, 1446, 1317, 1161, 748 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz, 1H), 8.31 (d, J=8.1 Hz, 1H), 7.90-7.88(m, 2H), 7.78 (d, J=8.3 Hz, 1H), 7.65 (d, J=5.9 Hz, 1H), 7.51-7.38 (m,7H), 5.96 (t, J=5.0 Hz, 1H), 4.67 (t, J=6.2 Hz, 2H), 3.01 (q, J=4.8 Hz,2H), 2.15 (m, 2H); APCI-MS m/z (rel intensity) 445 (MH⁺, 100); HRMS(+ESI) calcd for MNa⁺: 467.1042. found: 467.1047; HPLC purity: 95.8%(MeOH, 100%), 95.1% (MeOH—H₂O, 95:5).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)butyl)-benzenesulfonamide(93)

The crude product was eluted with EtOAc—CHCl₃ (3:7) to afford thedesired product as a red solid (57.2 mg, 89%): mp 184-186° C. IR (film)3222, 1700, 1652, 1614, 1551, 1507, 1427, 1324, 1164, 753 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.71 (d, J=8.0 Hz, 1H), 8.32 (d, J=8.0 Hz, 1H), 7.89(d, J=6.9 Hz, 2H), 7.75 (t, J=7.1 Hz, 1H), 7.65 (d, J=7.1 Hz, 1H),7.56-7.39 (m, 7H), 4.83 (t, J=6.2 Hz, 1H), 4.54 (t, J=7.5 Hz, 2H), 3.14(q, J=6.5 Hz, 2H), 1.98 (m, 2H), 1.78 (m, 2H); ESI-MS m/z (relintensity) 459 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 459.1379. found:459.1375; HPLC purity: 98.5% (MeOH, 100%), 98.3% (MeOH—H₂O, 90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)pentyl)-benzenesulfonamide(94)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (55.8 mg, 84%): mp 194-195° C. IR(film) 3284, 1728, 1699, 1662, 1609, 1548, 1504, 1446, 1326, 1261, 1161,758 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.36 (d,J=8.4 Hz, 1H), 7.87 (dd, J=1.5 and 5.4 Hz, 2H), 7.76 (td, J=1.3 and 7.0Hz, 1H), 7.65 (d, J=6.8 Hz, 1H), 7.57-7.38 (m, 7H), 4.66 (t, J=6.1 Hz,1H), 4.52 (t, J=7.5 Hz, 2H), 3.05 (q, J=6.3 Hz, 2H), 1.91 (m, 2H), 1.67(m, 2H), 1.58 (m, 2H); ESI-MS m/z (rel intensity) 473 (MH⁺, 100); HRMS(+ESI) calcd for MH⁺: 473.1535. found: 473.1532; HPLC purity: 99.5%(MeOH, 100%), 98.7% (MeOH—H₂O, 90:10).

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)hexyl)-benzenesulfonamide(95)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (57.8 mg, 91%): mp 150-152° C. IR(film) 3272, 1698, 1663, 1503, 1427, 1320, 1159, 756 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.35 (d, J=7.6 Hz, 1H), 7.89 (dd,J=6.7 and 1.6 Hz, 2H), 7.76 (t, J=8.2 Hz, 1H), 7.66 (d, J=6.9 Hz, 1H),7.58-7.41 (m, 7H), 4.56-4.48 (m, 3H), 3.03 (q, J=6.3 Hz, 2H), 1.88 (m,2H), 1.56-1.48 (m, 6H); ESI-MS m/z (rel intensity) 509 (MNa⁺, 100); HRMS(+ESI) calcd for MNa⁺: 509.1511. found: 509.1521; HPLC purity: 97.9%(MeOH, 100%), 97.4% (MeOH—H₂O, 90:10).

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)heptyl)-benzenesulfonamide(96)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired compound as an orange product (37.8 mg, 75%): mp 178-181° C. IR(film) 3281, 1698, 1669, 1608, 1548, 1504, 1446, 1324, 1160, 759 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.37 (d, J=8.0 Hz,1H), 7.88 (dd, J=0.92 and 6.8 Hz, 2H), 7.66 (t, J=6.7 Hz, 1H), 7.57 (d,J=6.9 Hz, 1H), 7.54-7.43 (m, 7H), 4.52 (t, J=6.8 Hz, 3H), 3.01 (q, J=6.6Hz, 2H), 1.87 (m, 2H), 1.49-1.25 (m, 8H); ESI-MS m/z (rel intensity) 501(MH⁺, 45); HRMS (+ESI) calcd for MNa⁺: 523.1668. found: 523.1672. Anal.calcd for C₂₉H₂₈N₂O₄S.0.7H₂O: C, 67.87; H, 5.77; N, 5.46. Found: C,67.50; H, 5.40; N, 5.37.

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)octyl)-benzenesulfonamide(97)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired compound as an orange product (36.1 mg, 72%): mp 136-140° C. IR(film) 3271, 1698, 1663, 1504, 1427, 1320, 1159, 756 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.36 (d, J=8.0 Hz, 1H), 7.88 (d,J=6.9 Hz, 2H), 7.70 (t, J=7.0 Hz, 1H), 7.66 (d, J=6.7 Hz, 1H), 7.61-7.38(m, 7H), 4.52 (t, J=7.8 Hz, 2H), 4.36 (m, 1H), 3.00 (q, J=6.8 Hz, 2H),1.91 (m, 2H), 1.47-1.25 (m, 10H); ESI-MS m/z (rel intensity) 1051(M₂Na⁺, 100); HRMS (+ESI) calcd for MH⁺: 515.2005. found: 515.2014; HPLCpurity: 99.4% (MeOH, 100%), 99.3% (MeOH—H₂O, 90:10).

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)nonyl)-benzenesulfonamide(98)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as an orange solid (58.2 mg, 78%): mp 162-164° C. ¹HNMR (300 MHz, CDCl₃) δ 8.73 (d, J=7.9 Hz, 1H), 8.36 (d, J=8.4 Hz, 1H),7.87 (d, J=7.0 Hz, 2H), 7.84-7.43 (m, 9H), 4.53 (t, J=8.1 Hz, 2H), 4.38(m, 1H), 2.99 (q, J=6.9 Hz, 2H), 1.89 (m, 2H), 1.57-1.25 (m, 12H);ESI-MS m/z (rel intensity) 551 (MNa⁺, 100); HRMS (+ESI) calcd for MH⁺:529.2161. found: 529.2159; HPLC purity: 100% (MeOH, 100%), 96.0%(MeOH—H₂O, 90:10).

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)decyl)-benzenesulfonamide(99)

The crude product was eluted with EtOAc—CHCl₃ (15:85) to afford thedesired product as an orange solid (54.6 mg, 88%): mp 128-130° C. IR(film) 3274, 1698, 1667, 1611, 1550, 1505, 1428, 1320, 1160, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.36 (d, J=8.2 Hz,1H), 7.88 (dd, J=1.5 and 5.3 Hz, 2H), 7.73 (dt, J=1.3 and 7.0 Hz, 1H),7.66 (d, J=6.2 Hz, 1H), 7.57-7.40 (m, 7H), 4.53 (t, J=7.6 Hz, 2H), 4.37(t, J=6.1 Hz, 1H), 2.99 (q, J=6.7 Hz, 2H), 1.90 (m, 2H), 1.52-1.24 (m,14H); APCI-MS m/z (rel intensity) 543 (MH⁺, 100); HRMS (+APCI) calcd forMNa⁺: 565.2137. found: 565.2142; HPLC purity: 100% (MeOH, 100%), 99.4%(MeOH—H₂O, 90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)undecyl)-benzenesulfonamide(100)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as a red solid (53.4 mg, 87%): mp 170-172° C. IR (film)3289, 1698, 1669, 1609, 1548, 1445, 1326, 1160, 760 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.4 Hz, 1H), 7.88 (dd,J=1.6 and 6.9 Hz, 2H), 7.76 (dt, J=1.2 and 7.1 Hz, 1H), 7.66 (d, J=6.2Hz, 1H), 7.61-7.39 (m, 7H), 4.53 (t, J=8.1 Hz, 2H), 4.38 (m, 1H), 2.99(q, J=6.9 Hz, 2H), 1.89 (m, 2H), 1.57-1.25 (m, 16H); ESI-MS m/z (relintensity) 557 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 557.2474. found:557.2477; HPLC purity: 98.8% (MeOH, 100%), 100% (MeOH—H₂O, 90:10).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)dodecyl)-benzenesulfonamide(101)

The crude product was eluted with EtOAc—CHCl₃ (7:3) to afford thedesired compound as an orange solid (62.8 mg, 86%): mp 128-130° C. IR(film) 3275, 1698, 1662, 1610, 1504, 1426, 1319, 1159, 756 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.35 (d, J=8.1 Hz, 1H), 7.87(d, J=7.0 Hz, 2H), 7.84-7.42 (m, 9H), 4.52 (t, J=7.6 Hz, 2H), 4.39 (m,1H), 2.98 (q, J=6.7 Hz, 2H), 1.89 (m, 2H), 1.59-1.21 (m, 12H); ESI-MSm/z (rel intensity) 571 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 571.2631.found: 571.2628. Anal. calcd for C₃₄H₃₈N₂O₄S: C, 71.55; H, 6.71; N,4.91. Found: C, 71.70; H, 6.82; N, 5.09.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)ethyl)-4-methylbenzenesulfonamide(102)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (57.4 mg, 84%): mp 272-274° C. IR(film) 3209, 1702, 1646, 1611, 1552, 1506, 1427, 1322, 1147, 763 cm⁻¹;₁H NMR (300 MHz, DMSO-d₆) δ 8.55 (d, J=8.0 Hz, 1H), 8.18 (d, J=7.6 Hz,1H), 8.01 (m, 1H), 7.83-7.76 (m, 2H), 7.58-7.48 (m, 6H), 7.26 (d, J=8.1Hz, 2H), 4.53 (t, J=7.0 Hz, 2H), 3.24 (t, J=6.7 Hz, 2H), 2.29 (s, 3H);ESI-MS m/z (rel intensity) 443 ([M-H]⁻¹, 100); HRMS (+ESI) calcd forMH⁺: 445.1222. found: 445.1220; HPLC purity: 98.6% (MeOH, 100%), 96.4%(MeOH—H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)-4-methylbenzenesulfonamide(103)

The crude product was eluted with EtOAc—CHCl₃ (3:7) to afford thedesired product as an orange solid (58.8 mg, 87%): mp 213-216° C. IR(film) 3294, 1705, 1638, 1609, 1548, 1501, 1422, 1327, 1163, 758 cm⁻¹;¹H NMR (300 MHz, DMSO-d₆) δ 8.58 (d, J=8.1 Hz, 1H), 8.21 (d, J=8.4 Hz,1H), 7.82-7.74 (m, 3H), 7.65-7.63 (m, 2H), 7.59-7.50 (m, 4H), 7.33-7.31(m, 2H), 4.49 (t, J=7.5 Hz, 2H), 2.93 (m, 2H), 2.33 (s, 3H), 1.92 (m,2H); ESI-MS m/z (rel intensity) 459 (MH⁺, 100); HRMS (+ESI) calcd forMH⁺: 459.1379. found: 459.1384; HPLC purity: 100% (MeOH, 100%), 99.4%(MeOH—H₂O, 90:10).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)butyl)-4-methylbenzenesulfonamide(104)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (59.7 mg, 90%): mp 190-192° C. IR(film) 3272, 1699, 1661, 1606, 1545, 1501, 1424, 1312, 1154, 759 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.71 (d, J=8.1 Hz, 1H), 8.32 (d, J=7.5 Hz,1H), 7.76-7.70 (m, 3H), 7.64 (d, J=6.9 Hz, 1H), 7.50-7.41 (m, 4H),7.30-7.26 (m, 2H), 4.76 (t, J=6.3 Hz, 1H), 4.54 (t, J=7.5 Hz, 2H), 3.11(q, J=6.5 Hz, 2H), 2.40 (s, 3H), 1.98 (m, 2H), 1.76 (m, 2H); ESI-MS m/z(rel intensity) 473 (MH⁺, 100); HRMS (+ESI) calcd for MNa⁺: 495.1355.found: 495.1362; HPLC purity: 96.4% (CH₃CN, 100%), 97.1% (CH₃CN—H₂O,90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)pentyl)-4-methylbenzenesulfonamide(105)

The crude product was eluted with EtOAc—CHCl₃ (15:85) to afford thedesired product as an orange solid (61.5 mg, 93%): mp 187-188° C. IR(film) 3273, 1694, 1671, 1609, 1548, 1504, 1426, 1325, 1159, 758 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.35 (d, J=7.4 Hz,1H), 7.76-7.70 (m, 3H), 7.65 (d, J=6.8 Hz, 1H), 7.50-7.38 (m, 4H),7.30-7.27 (m, 2H), 4.55 (m, 3H), 3.02 (q, J=6.4 Hz, 2H), 2.41 (s, 3H),1.90 (m, 2H), 1.65 (m, 2H), 1.54 (m, 2H); ESI-MS m/z (rel intensity) 487(MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 487.1692. found: 487.1688; HPLCpurity: 99.2% (MeOH, 100%), 98.1% (MeOH—H₂O, 90:10).

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)hexyl)-4-methylbenzenesulfonamide(106)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as a red solid (60.2 mg, 92%): mp 146-149° ° C. IR(film) 3583, 1688, 1652, 1608, 1500, 1419, 1314, 1151, 759 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.35 (d, J=8.1 Hz, 1H),7.76-7.71 (m, 3H), 7.66 (d, J=6.7 Hz, 1H), 7.50-7.40 (m, 4H), 7.30-7.27(m, 2H), 4.53 (m, 3H), 3.00 (q, J=6.2 Hz, 2H), 2.42 (s, 3H), 1.88 (m,2H), 1.53-1.48 (m, 6H); ESI-MS m/z (rel intensity) 501 (MH⁺, 100); HRMS(+ESI) calcd for MH⁺: 501.1848. found: 501.1860; HPLC purity: 96.4%(MeOH, 100%), 97.6% (MeOH—H₂O, 90:10).

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)heptyl)-4-methylbenzenesulfonamide(107)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as an orange solid (41.5 mg, 80%): mp 173-175° C. IR(film) 3275, 1696, 1663, 1609, 1575, 1549, 1503, 1426, 1321, 1158, 780cm⁻¹; ¹H NMR (300 MHz, CDCl₃) 8.72 (d, J=7.7 Hz, 1H), 8.36 (d, J=8.4 Hz,1H), 7.76-7.73 (m, 3H), 7.65 (d, J=5.9 Hz, 1H), 7.50-7.45 (m, 4H),7.31-7.29 (m, 2H), 4.49 (m, 3H), 2.96 (q, J=6.5 Hz, 2H), 2.42 (s, 3H),1.87 (m, 2H), 1.49-1.25 (m, 8H); ESI-MS m/z (rel intensity) 537 (MNa⁺,100); HRMS (+ESI) calcd for MNa⁺: 537.1824. found: 537.1819. Anal. calcdfor C₃₀H₃₀N₂O₄S.0.7H₂O: C, 68.34; H, 6.00; N, 5.31. Found: C, 67.97; H,5.75; N, 4.96.

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)octyl)-4-methylbenzenesulfonamide(108)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as an orange solid (36.0 mg, 70%): mp 136-139° C. IR(film) 3272, 1698, 1663, 1611, 1550, 1504, 1428, 1320, 1159, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.1 Hz,1H), 7.75-7.70 (m, 3H), 7.66 (d, J=6.9 Hz, 1H), 7.50-7.40 (m, 4H), 7.32(d, J=8.0 Hz, 2H), 4.53 (t, J=8.2 Hz, 2H), 4.31 (m, 1H), 2.97 (t, J=6.7,2H), 2.42 (s, 3H), 1.88 (m, 2H), 1.56-1.19 (m, 10H); ESI-MS m/z (relintensity) 529 (MH⁺, 78); HRMS (+ESI) calcd for MH⁺: 529.2161. found:529.2155; HPLC purity: 97.7% (MeOH, 100%), 96.7% (MeOH—H₂O, 90:10).

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)nonyl)-4-methylbenzenesulfonamide(109)

The crude product was eluted with EtOAc—CHCl₃ (7:3) to afford thedesired compound as an orange product (61.3 mg, 80%): mp 159-160° C. IR(film) 3276, 1769, 1698, 1663, 1610, 1549, 1504, 1427, 1320, 1158, 758cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.36 (d, J=7.4Hz, 1H), 7.75-7.70 (m, 3H), 7.66 (d, J=6.8 Hz, 1H), 7.49-7.39 (m, 4H),7.32-7.29 (m, 2H), 4.53 (t, J=7.9 Hz, 2H), 4.33 (t, J=7.1 Hz, 1H), 2.96(m, 2H), 2.42 (s, 3H), 1.88 (m, 2H), 1.51-1.26 (m, 12H); ESI-MS m/z (relintensity) 1107 (M₂Na⁺, 18); HRMS (+ESI) calcd for MH⁺: 543.2318. found:543.2307. Anal. calcd for C₃₂H₃₄N₂O₄S.0.2H₂O: C, 70.36; H, 6.35; N,5.13. Found: C, 70.22; H, 6.35; N, 4.97.

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)decyl)-4-methylbenzenesulfonamide(110)

The crude product was eluted with EtOAc—CHCl₃ (10:90) to afford thedesired product as a red solid (49.4 mg, 78%): mp 133-135° C. IR (film)3263, 1699, 1663, 1611, 1550, 1504, 1428, 1320, 1159, 758 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.36 (d, J=7.4 Hz, 1H),7.76-7.71 (m, 3H), 7.66 (d, J=6.3 Hz, 1H), 7.50-7.40 (m, 4H), 7.32-7.30(m, 2H), 4.53 (t, J=7.7 Hz, 2H), 4.33 (t, J=5.9 Hz, 1H), 2.97 (q, J=6.8Hz, 2H), 2.43 (s, 3H), 1.92 (m, 2H), 1.55-1.19 (m, 14H); APCI-MS m/z(rel intensity) 557 (MH⁺, 100); HRMS (+APCI) calcd for MH⁺: 557.2474.found: 557.2478; HPLC purity: 97.0% (MeOH, 100%), 98.8% (MeOH—H₂O,90:10).

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)undecyl)-4-methylbenzenesulfonamide(111)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as an orange solid (45.0 mg, 71.4%): mp 170-172° C. IR(film) 3289, 1698, 1669, 1609, 1575, 1548, 1505, 1445, 1326, 1160, 760cm⁻¹; ¹H-NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.0Hz, 1H), 7.76-7.70 (m, 3H), 7.65 (d, J=6.4 Hz, 1H), 7.49-7.40 (m, 4H),7.32-7.29 (m, 2H), 4.53 (t, J=7.5 Hz, 2H), 4.33 (br s, 1H), 2.97 (q,J=6.6 Hz, 2H), 2.43 (s, 3H), 1.90 (m, 2H), 1.58-1.23 (m, 16H); ESI-MSm/z (rel intensity) 571 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 571.2631.found: 571.2625; HPLC purity: 96.7% (MeOH, 100%), 96.7% (MeOH—H₂O,95:5).

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)dodecyl)-4-methylbenzenesulfonamide(112)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired compound as an orange solid (55 mg, 73%): mp 108-112° C. IR(film) 3275, 1698, 1666, 1611, 1550, 1504, 1428, 1320, 1160, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.2 Hz, 1H), 8.36 (d, J=7.0 Hz,1H), 7.75-7.63 (m, 4H), 7.49-7.29 (m, 6H), 4.53 (t, J=7.3 Hz, 2H), 4.27(m, 1H), 2.96 (q, J=6.7 Hz, 2H), 2.42 (s, 3H), 1.90 (m, 2H), 1.57-1.22(m, 18H); ESI-MS m/z (rel intensity) 607 (MNa⁺, 30); HRMS (+ESI) calcdfor MNa⁺: 607.2607. found: 607.2604. Anal. calcd for C₃₅H₄₀H₂O₄S: C,71.89; H, 6.89; N, 4.79. Found: C, 71.49; H, 6.97; N, 4.83.

N-(2-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)ethyl)-4-bromobenzenesulfonamide(113)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (64.2 mg, 82%): mp 260-263° C. IR(film) 3208, 1707, 1645, 1611, 1551, 1506, 1427, 1324, 1146, 827 cm⁻¹;¹H NMR (300 MHz, DMSO-d₆) δ 8.56 (d, J=8.0 Hz, 1H), 8.22 (m, 2H),7.80-7.69 (m, 4H), 7.64-7.61 (m, 2H), 7.56-7.48 (m, 4H), 4.54 (t, J=6.4Hz, 2H), 3.26 (t, J=6.7 Hz, 2H); ESI-MS m/z (rel intensity) 507/509([M-H]⁻, 78/100); HRMS (−ESI) calcd for [M-H]⁻: 507.0014. found:507.0017. HPLC purity: 97.7% (MeOH, 100%), 95.3% (MeOH—H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)-4-bromobenzenesulfonamide(114)

The crude product was eluted with EtOAc—CHCl₃ (9:1) to afford thedesired product as an orange solid (40.2 mg, 52%): mp 241-243° C. IR(film) 3303, 1762, 1696, 1653, 1609, 1549, 1505, 1427, 1325, 1166, 751cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.72 (d, J=8.1 Hz, 1H), 8.28 (d, J=7.6Hz, 1H), 7.79-7.74 (m, 3H), 7.66-7.39 (m, 7H), 6.11 (t, J=6.7 Hz, 1H),4.67 (t, J=6.0 Hz, 2H), 3.01 (q, J=6.6 Hz, 2H), 2.18 (m, 2H); ESI-MS m/z(rel intensity) 523/525 (MH⁺, 100/93); HRMS (+ESI) calcd for MH⁺:523.0327. found: 523.0335; HPLC purity: 95.4% (MeOH, 100%), 95.7%(MeOH—H₂O, 95:5).

N-(4-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)butyl)-4-bromobenzenesulfonamide(115)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as a red solid (63.7 mg, 84%): mp 176-177° C. IR (film)3257, 1698, 1663, 1611, 1576, 1503, 1427, 1332, 1163, 757 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.71 (d, J=8.1 Hz, 1H), 8.32 (d, J=8.0 Hz, 1H),7.76-7.70 (m, 3H), 7.67-7.63 (m, 3H), 7.49-7.40 (m, 4H), 4.98 (t, J=6.3Hz, 1H), 4.54 (t, J=7.3 Hz, 2H), 3.14 (q, J=6.4 Hz, 2 Hz), 1.99 (m, 2H),1.80 (m, 2H); ESI-MS m/z (rel intensity) 537/539 (MH⁺, 89/100); HRMS(+ESI) calcd for MH⁺: 537.0484. found: 537.0489; HPLC purity: 99.4%(MeOH, 100%), 98.1% (MeOH—H₂O, 90:10).

N-(5-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)pentyl)-4-bromobenzenesulfonamide(116)

The crude product was eluted with EtOAc—CHCl₃ (15:85) to afford thedesired product as an orange solid (68.0 mg, 91%): mp 169-171° C. IR(film) 3271, 1698, 1661, 1611, 1576, 1505, 1428, 1332, 1163, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=7.3 Hz,1H), 7.74-7.70 (m, 3H), 7.66-7.61 (m, 3H), 7.49-7.41 (m, 4H), 4.73 (t,J=6.0 Hz, 1H), 4.54 (t, J=7.2 Hz, 2H), 3.04 (q, J=6.4 Hz, 2 Hz), 1.92(m, 2H), 1.68 (m, 2H), 1.53 (m, 2H); ESI-MS m/z (rel intensity) 551/553(MH⁺, 100/98); HRMS (+ESI) calcd for MH⁺: 551.0640. found: 551.0652;HPLC purity: 98.3% (MeOH, 100%), 98.2% (MeOH—H₂O, 90:10).

N-(6-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)hexyl)-4-bromobenzenesulfonamide(117)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as a red solid (68.5 mg, 93%): mp 188-190° C. IR (film)3199, 1760, 1698, 1636, 1610, 1574, 1503, 1458, 1330, 1160, 757 cm⁻¹; ¹HNMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.35 (d, J=7.5 Hz, 1H),7.76-7.71 (m, 3H), 7.66-7.64 (m, 3H), 7.50-7.39 (m, 4H), 4.74 (t, J=6.2Hz, 1H), 4.54 (t, J=7.2 Hz, 2H), 3.03 (q, J=6.4 Hz, 2 Hz), 1.89 (m, 2H),1.57-1.48 (m, 6H); ESI-MS m/z (rel intensity) 565/567 (MH⁺, 91/100);HRMS (+ESI) calcd for MNa⁺: 587.0616. found: 587.0610; HPLC purity:96.6% (MeOH, 100%), 98.2% (MeOH—H₂O, 90:10).

N-(7-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)heptyl)-4-bromobenzenesulfonamide(118)

The crude product was eluted with EtOAc—CHCl₃ (5:95) to afford thedesired product as a red solid (70.5 mg, 96%): mp 160-161° C. IR (film)3290, 1699, 1661, 1610, 1550, 1503, 1427, 1320, 1161, 757 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.36 (d, J=7.7 Hz, 1H),7.76-7.70 (m, 3H), 7.66-7.63 (m, 3H), 7.50-7.39 (m, 4H), 4.69 (t, J=6.1Hz, 1H), 4.51 (t, J=7.7 Hz, 2H), 3.01 (q, J=6.7 Hz, 2 Hz), 1.90 (m, 2H),1.53-1.36 (m, 8H); ESI-MS m/z (rel intensity) 579/581 (MH⁺, 92/100);HRMS (+ESI) calcd for MNa⁺: 579.0953. found: 579.0959; HPLC purity:98.3% (MeOH, 100%), 98.9% (MeOH—H₂O, 90:10).

N-(8-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)octyl)-4-bromobenzenesulfonamide(119)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (67.6 mg, 78%): mp 119-121° C. IR(film) 3272, 1698, 1662, 1611, 1550, 1504, 1428, 1331, 1163, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.73 (d, J=8.1 Hz, 1H), 8.36 (d, J=8.4 Hz,1H), 7.74-7.71 (m, 3H), 7.67-7.64 (m, 3H), 7.47-7.43 (m, 4H), 4.53 (t,J=7.7 Hz, 2H), 4.43 (t, J=6.4 Hz, 1H), 3.00 (q, J=6.8 Hz, 2H), 1.91 (m,2H), 1.55-1.20 (m, 10H); ESI-MS m/z (rel intensity) 593/595 (MH⁺,100/91); HRMS (+ESI) calcd for MH⁺: 593.1110. found: 593.1105. Anal.calcd for C₃₀H₂₉BrN₂O₄S: C, 60.71; H, 4.92; N, 4.72. Found: C, 60.69; H,5.01; N, 4.67.

N-(9-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)nonyl)-4-bromobenzenesulfonamide(120)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (52.2 mg, 61%): mp 143-146° C. IR(film) 3288, 1697, 1662, 1610, 1550, 1504, 1427, 1320, 1162, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.1 Hz, 1H), 8.35 (d, J=8.2 Hz,1H), 7.74-7.71 (m, 3H), 7.66-7.63 (m, 3H), 7.49-7.40 (m, 4H), 4.53 (t,J=6.4 Hz, 3H), 2.99 (q, J=6.8 Hz, 2H), 1.91 (m, 2H), 1.58-1.25 (m, 12H);ESI-MS m/z (rel intensity) 607/609 (MH⁺, 88/100); HRMS (+ESI) calcd forMH⁺: 607.1266. found: 607.1263. Anal. calcd for C₃₁H₃₁BrN₂O₄S: C, 61.28;H, 5.14; N, 4.61. Found: C, 61.22; H, 5.16; N, 4.57.

N-(10-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)decyl)-4-bromobenzenesulfonamide(121)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (65.2 mg, 77%): mp 160-163° C. IR(film) 3262, 1696, 1658, 1609, 1548, 1503, 1426, 1321, 1158, 753 cm⁻¹;¹H NMR (500 MHz, CDCl₃) δ 8.72 (d, J=8.0 Hz, 1H), 8.35 (d, J=8.0 Hz,1H), 7.74-7.71 (m, 3H), 7.66-7.64 (m, 3H), 7.48-7.40 (m, 4H), 4.52 (t,J=7.0 Hz, 2H), 4.40 (m, 1H), 2.97 (q, J=6.5 Hz, 2H), 1.91-1.88 (m, 2H),1.54-1.22 (m, 14H); ESI-MS m/z (rel intensity) 621/623 (MH⁺, 100/99.8);HRMS (+ESI) calcd for MH⁺: 621.1423. found: 621.1430. Anal. calcd forC₃₂H₃₃BrN₂O₄S: C, 61.83; H, 5.35; N, 4.51. Found: C, 61.95; H, 5.40; N,4.71.

N-(11-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)undecyl)-4-bromobenzenesulfonamide(122)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (60.6 mg, 86%): mp 150-153° C. IR(film) 3310, 1705, 1661, 1608, 1548, 1504, 1422, 1328, 1156, 757 cm⁻¹;¹H-NMR (300 MHz, CDCl₃) 8.73 (d, J=8.0 Hz, 1H), 8.36 (d, J=8.2 Hz, 1H),7.75-7.71 (m, 3H), 7.66-7.61 (m, 3H), 7.49-7.38 (m, 4H), 4.54 (t, J=7.5Hz, 2H), 4.40 (t, J=6.1 Hz, 1H), 2.99 (q, J=6.8 Hz, 2H), 1.90 (m, 2H),1.56-1.23 (m, 16H); ESI-MS m/z (rel intensity) 635/637 (MH⁺, 100/92);HRMS (+ESI) calcd for MH⁺: 635.1579. found: 635.1584. Anal. calcd forC₃₃H₃₅BrN₂O₄S: C, 62.36; H, 5.55; N, 4.41. Found: C, 62.15; H, 5.62; N,4.44.

N-(12-(5,11-Dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)dodecyl)-4-bromobenzenesulfonamide(123)

The crude product was eluted with EtOAc—CHCl₃ (6:4) to afford thedesired product as an orange solid (65.3 mg, 78%): mp 118-120° C. IR(film) 3275, 1698, 1664, 1611, 1550, 1504, 1428, 1332, 1164, 757 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ 8.72 (d, J=8.3 Hz, 1H), 8.35 (d, J=8.1 Hz,1H), 7.73-7.63 (m, 6H), 7.49-7.40 (m, 4H), 4.53 (t, J=7.8 Hz, 2H), 4.41(t, J=5.9 Hz, 1H), 2.99 (q, J=6.7 Hz, 2H), 1.90-1.87 (m, 2H), 1.58-1.21(m, 18H); ESI-MS m/z (rel intensity) 649/651 (MH⁺, 100/80); HRMS (+ESI)calcd for MH⁺: 649.1736. found: 649.1728. Anal. calcd forC₃₄H₃₇BrN₂O₄S.0.5H₂O: C, 62.00; H, 5.82; N, 4.25. Found: C, 61.71; H,5.62; N, 4.15.

General Procedure for the Preparation of Indenoisoquinoline Sulfonamides132-136

Indenoisoquinoline salts 127-131 (50 mg, 0.088-0.102 mmol), preparedpreviously via the reported literature procedures, were dissolved inCHCl₃ (15 mL), Et₃N (2 equiv) in CHCl₃ (1 mL) was added, and thesolutions were stirred at room temperature for 5 min. Benzenesulfonylchloride (2 equiv) was added. The mixtures were heated at reflux for 3h, and then washed with sat. NaHCO₃ (50 mL) and brine (50 mL). Theorganic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated, adsorbed onto SiO₂, and purified by flash columnchromatography (SiO₂), eluting with EtOAc—CHCl₃ to provide theindenoisoquinoline sulfonamide products 132-136 in high purity.

N-(3-(3-Nitro-5,11-dioxo-9-phenyl-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-propyl)benzenesulfonamide(132)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (22.9 mg, 37%): mp 246-247° C. IR(film) 3318, 1706, 1658, 1615, 1553, 1501, 1448, 1383, 1336, 1153, 749cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.79 (d, J=2.2 Hz, 1H), 8.66 (d, J=9.0Hz, 1H), 8.54 (dd, J=6.5 and 2.4 Hz, 1H), 7.89-8.75 (m, 8H), 7.61-7.45(m, 6H) 4.50 (m, 2H), 3.02 (m, 2H), 1.97 (m, 2H); ESI-MS m/z (relintensity) 566 (MH⁺, weak); HRMS (+ESI) calcd for MH⁺: 566.1386. found:566.1378; HPLC purity: 100% (MeOH, 100%), 99.1% (MeOH—H₂O, 90:10).

N-(3-(5,11-Dioxo-5H-[1,3]dioxolo[4,5-g]indeno[1,2-c]isoquinolin-6(11H)-yl)-propyl)benzenesulfonamide(133)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (51.3 mg, 81%): mp 225-226° C. IR(film) 3223, 1700, 1636, 1608, 1562, 1499, 1467, 1329, 1174, 765 cm⁻¹;¹H NMR (300 MHz, DMSO-d₆) δ 7.88-7.76 (m, 4H), 7.60 (d, J=5.3 Hz, 1H),7.58-7.47 (m, 7H), 6.19 (s, 2H), 4.43 (t, J=7.3 Hz, 2H), 2.95 (q, J=6.0Hz, 2H), 1.90 (m, 2H); APCI-MS m/z (rel intensity) 489 (MH⁺, 100); HRMS(+APCI) calcd for MNa⁺: 511.0940. found: 511.0949; HPLC purity: 100%(MeOH, 100%), 99.9% (MeOH:H₂O, 90-10).

Methyl2,3-Dimethoxy-5,11-dioxo-6-(3-(phenylsulfonamido)propyl)-6,11-dihydro-5H-indeno[1,2-c]isoquinoline-9-carboxylate(134)

The crude product was eluted with EtOAc—CHCl₃ (4:6) to afford thedesired product as a deep purple solid (53.7 mg, 88%): mp 249-251° C. IR(film) 3244, 1727, 1645, 1611, 1554, 1511, 1482, 1321, 1259, 1161, 802,764 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.15-8.12 (m, 2H), 8.07 (s, 1H),7.92 (d, J=7.1 Hz, 2H), 7.63 (s, 1H), 7.56-7.45 (m, 4H), 6.04 (t, J=6.4Hz, 1H), 4.65 (t, J=6.1 Hz, 2H), 4.07 (s, 3H), 4.03 (s, 3H), 3.97 (s,3H), 3.08 (q, J=6.2 Hz, 2H), 2.12 (m, 2H); APCI-MS m/z (rel intensity)563 (MH⁺, 100); HRMS (+APCI) calcd for MNa⁺: 585.1308. found: 585.1302;HPLC purity: 100% (MeOH, 100%), 99.9% (MeOH:H₂O, 90-10).

N-(3-(3-Nitro-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)propyl)-benzenesulfonamide(135)

The crude product was eluted with EtOAc—CHCl₃ (2:8) to afford thedesired product as an orange solid (28.6 mg, 46%): mp 258-259° C. IR(film) 3214, 1699, 1656, 1613, 1553, 1503, 1453, 1423, 1381, 1324, 1259,1158, 802, 746 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.88 (d, J=2.4 Hz, 1H),8.74 (d, J=8.9 Hz, 1H), 8.60 (dd, J=6.5 and 2.4 Hz, 1H), 7.91 (t, J=8.0Hz, 2H), 7.82 (dd, J=6.7 and 1.5 Hz, 2H), 7.66-7.57 (m, 6H), 4.51 (t,J=8.0 Hz, 2H), 3.01 (q, J=6.2 Hz, 2H), 1.96 (m, 2H); APCI-MS m/z (relintensity) 490 (MH⁺, 100); HRMS (+APCI) calcd for MH⁺: 490.1073. found:490.1070; HPLC purity: 97.1% (MeOH, 100%), 100% (MeOH—H₂O, 90:10).

N-(3-(2,3-Dimethoxy-5,11-dioxo-5H-indeno[1,2-c]isoquinolin-6(11H)-yl)-propyl)benzenesulfonamide(136)

The crude product was eluted with EtOAc—CHCl₃ (4:6) to afford thedesired product as an orange solid (58.9 mg, 94%): mp 249-250° C. IR(film) 3166, 1701, 1626, 1612, 1589, 1554, 1513, 1478, 1426, 1335, 1260,1171, 1093, 1021, 789 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.12 (s, 1H), 7.91(dd, J=6.8 and 1.6 Hz, 2H), 7.64 (s, 1H), 7.60 (d, J=6.6 Hz, 1H),7.52-7.37 (m, 6H), 6.12 (t, J=6.9 Hz, 1H), 4.65 (t, J=5.9 Hz, 2H), 4.07(s, 3H), 4.03 (s, 3H), 3.05 (q, J=6.3 Hz, 2H), 2.13 (m, 2H); ESI-MS m/z(rel intensity) 505 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 505.1433.found: 505.1423; HPLC purity: 100% (MeOH, 100%), 100% (MeOH—H₂O, 90:10).

General Procedure for the Preparation of Bisindenoisoquinolines 140-142

Diamines 137-139 (100 mg, 1 equiv) were dissolved in CHCl₃ (10 mL) andadded to a solution of lactone 11 (2 equiv) in CHCl₃ (20 mL). Themixtures were stirred at reflux for 16 h, and then concentrated,adsorbed onto SiO₂, and purified with flash column chromatography(SiO₂), eluting with CHCl₃ to afford the products 140-142 as orange orred solids.

6,6′-(Decane-1,10-diyl)bis(5H-indeno[1,2-c]isoquinoline-5,11[6H]-dione)(140)

The general procedure provided the product as an orange solid (223 mg,64%): mp 201-202° C. IR (film) 1762, 1657, 1609 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 8.72 (d, J=8.1 Hz, 2H), 8.35 (d, J=8.2 Hz, 2H), 7.75 (d, J=8.3Hz, 2H), 7.64 (d, J=6.4 Hz, 2H), 7.49-7.39 (m, 8H), 4.53 (t, J=7.9 Hz,4H), 1.90 (m, 4H), 1.57 (m, 4H), 1.54-1.38 (m, 8H); ESI-MS m/z (relintensity) 633 (MH⁺, 57); HRMS (+ESI) calcd for MH⁺: 633.2753. found:633.2763. Anal. calcd for C₄₂H₃₆N₂O₄: C, 79.72; H, 5.73; N, 4.43. Found:C, 79.53; H, 5.76; N, 4.39.

6,6′-(Undecane-1,11-diyl)bis(5H-indeno[1,2-c]isoquinoline-5,11[6H]-dione)(141)

The general procedure provided the product as an orange solid (223 mg,64%): mp 201-202° C. IR (film) 1764, 1736, 1702, 1655 cm⁻¹; ¹H NMR (300MHz, CDCl₃) δ 8.71 (d, J=8.1 Hz, 2H), 8.35 (d, J=7.7 Hz, 2H), 7.82 (d,J=8.2 Hz, 2H), 7.64 (d, J=6.5 Hz, 2H), 7.48-7.38 (m, 8H), 4.52 (t, J=8.1Hz, 4H), 1.92 (m, 4H), 1.58 (m, 4H), 1.41-1.33 (m, 10H); ESI-MS m/z (relintensity) 647 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 647.2910. found:647.2908. Anal. calcd for C₄₃H₃₈N₂O₄: C, 79.85; H, 5.92; N, 4.33. Found:C, 79.80; H, 5.99; N, 4.29.

6,6′-(Dodecane-1,12-diyl)bis(5H-indeno[1,2-c]isoquinoline-5,11[6H]-dione)(142)

The general procedure provided the product as a red solid (135 mg, 82%):mp 226-228° C. IR (film) 1694, 1660, 1609 cm⁻¹; ¹H NMR (300 MHz, CDCl₃):δ8.71 (d, J=8.0 Hz, 2H), 8.35 (d, J=8.1 Hz, 2H), 7.74 (t, J=7.5 Hz, 2H),7.64 (d, J=6.9 Hz, 2H), 7.48-7.37 (m, 8H), 4.52 (t, J=7.9 Hz, 4 Hz),1.90 (m, 4H), 1.53 (m, 4H), 1.40-1.31 (m, 12H); ESI-MS m/z (relintensity) 661 (MH⁺, 100); HRMS (+ESI) calcd for MH⁺: 661.3066. found:661.3054; HPLC purity: 99.7% (CH₃CN, 100%), 97.0% (CH₃CN—H₂O, 95:5).

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-(6-propyl-tert-BOCamino)}propane(144)

Tetramine 143 (100 mg, 0.53 mmol) was diluted in CHCl₃ (10 mL) and addedto a solution of lactone 11 (276 mg, 1.12 mmol) in CHCl₃ (40 mL). Thereaction mixture was stirred at reflux for 72 h. Et₃N (270 mg, 2.66mmol) and Boc₂O (360 mg, 1.65 mmol) were then added to the cooledmixture, and stirring continued at room temperature for 16 h. Themixture was concentrated, adsorbed on to SiO₂, and purified with flashcolumn chromatography (SiO₂), eluting with EtOAc-hexane (2:8) and thenwith MeOH—CHCl₃ (3:97) to provide the product as an orange solid (352mg, 78%): mp 84-86° C. (lit. 86-88° C.). ¹H NMR (CDCl₃) δ 8.63 (d, J=7.9Hz, 2H), 8.24 (d, J=7.6 Hz, 2H), 7.65 (t, J=7.3 Hz, 2H), 7.55 (d, J=6.9Hz, 2H), 7.41-7.30 (m, 8H), 4.49 (br s, 4H), 3.44 (br s, 4H), 3.27 (t,J=6.3 Hz, 4H), 2.08 (brs, 4H), 1.86 (br s, 2H), 1.41 (br s, 18H).

Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propyl-amino}propaneBis(trifluoroacetate) (145)

Boc-protected bis(indenoisoquinoline) 144 (352 mg, 0.41 mmol) wasdiluted in CF₃COOH (30 mL) and the mixture was stirred at roomtemperature for 2 h. The reaction mixture was concentrated, and theresultant solid was triturated with chloroform and filtered to providethe product as an orange solid (247 mg, 93%): mp 224-226° C. (lit.225-227° C.). ¹H NMR (DMSO-d₆) δ8.59-8.57 (m, 6H), 8.22 (d, J=8.1 Hz,2H), 7.86-7.78 (m, 4H), 7.62-7.49 (m, 8H), 4.60 (t, J=6.4 Hz, 4H), 3.08(br s, 4H), 2.96 (br s, 4H), 2.15 (br s, 4H), 1.90 (m, 2H); HPLC purity:97.7% (MeOH—H₂O, 80:20), 97.6% (MeOH—H₂O, 70:30).

Biological Tests

Topoisomerase I-Mediated DNA Cleavage Reactions.

Human recombinant Top1 was purified from baculovirus as previouslydescribed. DNA cleavage reactions were prepared as previously reportedwith the exception of the DNA substrate. Briefly, a 117-bp DNAoligonucleotide (Integrated DNA Technologies) encompassing thepreviously identified Top1 cleavage sites in the 161-bp fragment frompBluescript SK(−) phagemid DNA was employed. This 117-bp oligonucleotidecontains a single 5′-cytosine overhang, which was 3′-end-labeled byfill-in reaction with [α-³²P]dGTP in React 2 buffer (50 mM Tris-HCl, pH8.0, 100 mM MgCl₂, 50 mM NaCl) with 0.5 unit of DNA polymerase I (Klenowfragment, New England BioLabs). Unincorporated [³²P]dGTP was removedusing mini Quick Spin DNA columns (Roche, Indianapolis, Ind.), and theeluate containing the 3′-end-labeled DNA substrate was collected.Approximately 2 nM radiolabeled DNA substrate was incubated withrecombinant Top1 in 20 μL of reaction buffer [10 mM Tris-HCl (pH 7.5),50 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, and 15 μg/mL BSA] at 25° C. for 20min in the presence of various concentrations of compounds. Thereactions were terminated by adding SDS (0.5% final concentration)followed by the addition of two volumes of loading dye (80% formamide,10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1%bromphenol blue). Aliquots of each reaction mixture were subjected to20% denaturing PAGE. Gels were dried and visualized by using aphosphoimager and ImageQuant software (Molecular Dynamics). Forsimplicity, cleavage sites were numbered as previously described in the161-bp fragment.

Gel-Based Assay Measuring the Inhibition of Recombinant Tdp1.

A 5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a3′-phosphotyrosine (N14Y) was generated as described by Dexheimer et al.The DNA substrate was then incubated with 5 pM recombinant Tdp1 in theabsence or presence of inhibitor for 15 min at room temperature in abuffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mMDTT, 40 μg/ml BSA and 0.01% Tween-20. Reactions were terminated by theaddition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mMEDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue].Samples were subjected to a 16% denaturing PAGE and gels were exposedafter drying to a PhosphorImager screen (GE Healthcare). Gel images werescanned using a Typhoon 8600 (GE Healthcare) and densitometric analyseswere performed using the ImageQuant software (GE Healthcare).

Gel-based Assay Measuring the Inhibition of Endogenous Human Tdp1 inWhole Cell Extract

1×10⁷ DT40 knockout cells for chicken Tdp1 and complemented with humanTdp1 were collected, washed and centrifuged. Cell pellet was thenresuspended with 100 μL of CellLytic M Cell Lysis Reagent (SIGMA-AldrichC2978). After 15 min, the lysate was centrifuged at a 12,000 g for 10min and the supernatant was transferred to a new tube. Proteinconcentration was determined using a nanodrop spectrophotometer(Invitrogen) and the whole cell extract was stored at −80° C. The5′-[³²P]-labeled single-stranded N14Y DNA oligonucleotide containing a3′-phosphotyrosine (see above) was incubated with 1-5 μg/ml of wholecell extract in the absence or presence of inhibitor for 15 min at roomtemperature in the same assay buffer used for recombinant Tdp1 (seesection above). Reactions were then treated similarly to the recombinantenzyme containing samples (see section above).

All compounds were first tested in gel based assays for Tdp1 inhibitionusing recombinant (rec.) human Tdp1 and only the active compounds weretested for human Tdp1 inhibition in whole cell extract (WCE). Furamidinewas included in all experiments as positive control.

Determination of the Mechanism of Inhibition of Compound 70.

Mechanistic characterization of compound 70 was carried out using a FRETassay employing a custom designed substrate (Bermingham et al.manuscript in preparation). The assay followed the real-time observationof reaction timecourse data, permitting an accurate measure of thereaction rate in the presence of an inhibitor. The mechanism ofinhibition of 70 was observed by measuring the rate of the Tdp1catalyzed reaction under a matrix of varying substrate and inhibitorconcentrations. In the assay, Tdp1 FRET substrate was present as adilution series ranging 2.25 μM to 0.035 μM over eight 2-fold steps.Compound 70 was present at three concentrations equaling 0.33×IC₅₀,1.0×IC₅₀ and 3.0×IC₅₀. A “no inhibitor” sample was also included, toallow measurement of the rate of reaction in the absence of inhibition.

Immediately prior to executing the assay, stocks of Tdp1 enzyme, Tdp1FRET substrate and compound 70 were created at 3× their final desiredassay concentrations. 5 μL of compound 70 stock dilutions were combinedwith 5 μL of a 1.5 nM Tdp1 stock in appropriate wells in a low volume384 well plate (Greiner #784900. Greiner, Monroe, N.C.) and allowed toincubate on ice for 1 hour to ensure complete binding equilibrium. Afterincubation, 5 μL of the 3×Tdp1 FRET substrate dilution series was addedto the plate to initiate the reaction. The assay plate was placed in aTecan Safire plate reader (Tecan US, Durham, N.C.) and timecourse dataobserved for 1 h at excitation and emission wavelengths of 525 nm (bandwidth=5) and 545 nm (band width=5) respectively for all wells. The finalassay volume was 15 μL, with Tdp1 present at a final fixed concentrationof 500 pM for all wells. Experimental data was plotted as reaction rateversus substrate concentration for all inhibitor concentrations andanalyzed using models for competitive, non-competitive (pure and mixed)and uncompetitive inhibition (Equations 1a-d respectively) usingGraphPad Prism (Graphpad, La Jolla, Calif.). To identify the mostappropriate mechanistic model describing the inhibition data, Akaike'sInformation Criterion (Akaike, H., 1973). Information theory and anextension of the maximum likelihood principle in Second InternationalSymposium on Information Theory (Csaki, B. N. P. a. F. ed., Budapest:Akademiai Kiado) was employed.

Equations

A. Competitive Inhibition

$v = \frac{V_{\max{\lbrack s\rbrack}}}{\lbrack S\rbrack + {K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)}}$Where V_(max) is maximum reaction velocity, [S] is the substrateconcentration, K_(m) is the Michaelis constant, and K_(i) is theinhibition constant.B. Pure Noncompetitive Inhibition

$v = \frac{V_{\max{\lbrack s\rbrack}}}{\left( {\lbrack S\rbrack + K_{m}} \right)\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)}$Where K_(i) is the inhibition constant in the presence or absence ofsubstrate.C. Mixed Noncompetitive Inhibition

$v = \begin{matrix}V_{\max{\lbrack s\rbrack}} \\{{\lbrack S\rbrack\left( {1 + \frac{\lbrack I\rbrack}{K_{ies}}} \right)} + {K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{ie}}} \right)}}\end{matrix}$Where K_(ie) is the inhibition constant for binding to the enzyme in theabsence of substrate, and K_(ies) the inhibition constant for binding tothe ES complex.D. Uncompetitive Inhibition

$v = \frac{V_{\max{\lbrack s\rbrack}}}{{\lbrack S\rbrack\left( {1 + \frac{\lbrack I\rbrack}{K_{ies}}} \right)} + K_{m}}$Where K_(ies) is the inhibition constant for binding to the ES complex.

Surface Plasmon Resonance Analysis

Binding experiments were performed on a Biacore T100 instrument (GE,Piscataway N.J.). Tdp1 was amine coupled to a CM5 sensor chip (GEHealthcare, Piscataway N.J.). Coupling reagents[N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide] (EDC),N-hydroxysuccinimide (NHS) and ethanolamine were purchased from GEHealthcare, (Piscataway N.J.). Neutravidin was obtained from Pierce. Inorder to protect the amine groups within the active site frommodification, Tdp1 was bound with a 14-base oligonucleotide beforecoupling to the surface. Specifically, 1 μM Tdp1 was incubated with 2 μMof a 14-base oligonucleotide containing a phosphate group at the 3′-end(GATCTAAAAGACTT) (SEQ ID NO: 1) in 10 mM sodium acetate pH 4.5 for 20min. The CM5 chip surface was activated for 7 min with 0.1 M NHS and 0.4M EDC at a flow rate of 20 μL/min and Tdp1-oligonucleotide mixture wasinjected until approximately 4000 RU's was attached. Activated aminegroups were quenched with an injection of 1 M solution of ethanolaminepH 8.0 for 7 min. Any bound oligonucleotide was removed by washing thesurface with 1 M NaCl. A reference surface was prepared in the samemanner without coupling of Tdp1. Compound 70 was diluted into runningbuffer [10 mM Hepes, 150 mM NaCl, 0.01% tween 20 (v/v), 5% DMSO (v/v) pH7.5] and injected over all flow cells at 30 μL/min at 25° C. Followingcompound injections, the surface was regenerated with a 30 secondinjection 1 M NaCl, a 30 second injection of 50% DMSO (v/v) and a 30second running buffer injection. Each cycle of compound injection wasfollowed by buffer cycle for referencing purposes. A DMSO calibrationcurve was included to correct for refractive index mismatches betweenthe running buffer and compound dilution series.

(B) J. Med. Chem. 2013, 56, 182-200

The phospholipases are a heterogeneous family of enzymes that catalyzethe hydrolysis of phosphodiester bonds. One member of this family,tyrosyl-DNA-phosphodiesterase I (Tdp1), catalyzes the hydrolysis of3′-phosphotyrosyl linkers. This enzyme is unique because its catalyticsite possesses two histidine and two lysine residues but lacks theaspartate residue that is found in the other members of the family. WhenDA topoisomerase I (Top1) nicks double-stranded DNA, a covalent Top1-DNAcomplex is made. The formed 3′-adducts must be removed by Tdp1 in orderto repair damaged DNA in stalled Top1-DNA complexes in which the normalDNA religation reaction has not occurred. The enzyme mechanism isbelieved to occur in two sequential steps. The first step consists ofthe nucleophilic attack by His263 on the phosphorous atom linked to theoxygen of the Top1 catalytic residue Tyr723 at the 3′-end of DNA (FIG.2). The function of the Lys265 and Lys495 residues found in thecatalytic site is to coordinate the oxygen atoms of the phosphate group,which enables the covalent linkage of His263 to the 3′-phosphate end ofthe DNA. In the second step, this intermediate is hydrolyzed by aHis493-activated water molecule. The overall reaction frees the tyrosineresidue and affords a DNA strand that has a 3′-phosphate end. Theprocess of DNA restoration is then finished by DNA polymerases and DNAligases. The role of Tdp1 is to hydrolyze phosphotyrosyl-DNA linkages indenatured or proteolytically degraded Top1-DNA complexes.

One approach to chemotherapy is based on forming lesions in tumor DNA.Thus, stalled Top1-DNA complexes can also arise when Top1 poisons, suchas camptothecins (CPTs) or indenoisoquinolines, are used for cancertherapy. The function of the CPTs is to stabilize the DNA-Top1 complexand inhibit the religation process. As a consequence, DNA replicationforks collide with drug-stabilized complexes causing double-stranded DNAbreaks that ultimately result in tumor cell death. It is believed thatTdp1 may be responsible for the drug resistance of some cancers byvirtue of repairing DNA lesions caused by CPTs. This hypothesis isfurther supported by the fact that hypersensitivity to CPTs is observedwhen Tdp1 is inactivated in DNA repair- or checkpoint-deficient yeast.

Given the relationship between Top1 and Tdp1, inhibitors of Tdp1 couldpotentiate the effects of Top1 poisons. Although the association betweenthese enzymes makes Tdp1 an attractive target for cancer treatment,there are few known inhibitors in the literature. Moreover, thesecompounds show weak inhibitory activity with IC₅₀'s usually in themicromolar range. A recent publication, (A) above, reported thatindenoisoquinolines bearing three or four methylene units on theamine-substituted side chain are good Tdp1 inhibitors. The bindinginteractions between Tdp1 and indenoisoquinoline 1 have previously beencharacterized using surface plasmon resonance (SPR) and fluorescenceresonance energy transfer (FRET). During the previous studies, it wasconcluded that the sulfonate substituent, key for the activity ofcompounds such as 2, did not provide any advantage when present on theside chain attached to the lactam of indenoisoquinolines.

This paper expands the previously reported SAR studies regarding Tdp1inhibitors and introduces novel indenoisoquinolines that are activeagainst this target. Additionally, compounds with and without dualactivity against both Tdp1 and Top1 enzymes are presented.

Chemistry

It is known that the Tdp1 active site possesses two lysine (265 and 495)and two histidine (263 and 493) residues. Some Tdp1 inhibitors havefunctional groups (e.g. carbonyls) that can hydrogen bond with theseresidues, and a lipophilic core that interacts with the hydrophobicregion (Ala520, Phe259, Gly260, Tyr261, etc.) of the active site.Indenoisoquinolines with an ester moiety might therefore be inhibitorsof the enzyme. The partially negatively charged carbonyl oxygen mayinteract with some of the charged polar residues in the binding pocketwhile the indenoisoquinoline aromatic system might sit in thehydrophobic portion of the cavity. In order to build on the previouswork, an ester moiety was therefore added to3-amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3) asshown in Scheme B1.

Briefly, the amino group of 3 was reacted with methyl oxalyl chloride(4) to provide compound 9 that was hydrolyzed, under basic conditions,to yield 14. Compound 9 was inactive but 14 showed modest Tdp1inhibitory activity with and IC₅₀ of 61.7 μM. Therefore, compounds 5-8with several methylene units between the acyl chloride and the estersfunctionalities were reacted with 3 to afford compounds 10-13. The ester10 was converted to the acid derivative 15.

Compound 16 was reacted with acyl chlorides 4-8 to produceindenoisoquinolines 17-21, which were converted to the amines 22-26 bydeprotection under acidic conditions (Scheme B2).

The acids 32-36 were prepared from intermediates 17-21 (Scheme B3) byhydrolysis under basic conditions followed by acid treatment.

Also, precursors 3 and 16 were reacted with ethyl glyoxylate using theBorch reduction to give esters 38 and 39 (Scheme B4) that were furtherhydrolyzed to acids 41 and 43. Compound 40 was prepared by deprotectionof 39 (Scheme B4).

A set of 3-nitroindenoisoquinolines was prepared because a high-prioritygoal was to synthesize dual Tdp1-Top1 inhibitors, and it is wellestablished that a 3-nitro substituent increases potency vs Top1.Compound 51 (Scheme B5) was prepared using the Schiff base-homophthalicanhydride condensation approach. Briefly, Schiff base 46, obtained bythe condensation of meta-methoxybenzaldehyde (44) and 3-bromopropylaminehydrobromide (45), was reacted with 5-nitrohomophthalic anhydride (47)to provide acid 48. This compound was subjected to Friedel-Craftsconditions to afford indenoisoquinoline 49, followed by halidedisplacement with azide to provide 50. Subsequent reduction of 50 underStaudinger conditions gave indenoisoquinoline 51.

Precursor 49 was treated with imidazole or morpholine to affordcompounds 52 and 53 (Scheme B6).

Previously reported compounds 54-63 from our existing library ofcompounds prepared to investigate Top1 inhibition, were also evaluatedin order to identify key features for Tdp1 inhibitory activity.

Additionally, new indenoisoquinolines bearing nitrile and iodosubstituents on the A ring were synthesized. There are not manysubstitutions reported in the literature on this side of theindenoisoquinoline system; thus, the current study provided a primeopportunity to expand the substitution pattern on the A ring. Moreover,given the fact that the methoxy or methylenedioxy substituents have beenshown to improve Top1 activity, these substituents were added to some ofthe new compounds. Isochromenone 68 (Scheme B7) was prepared bycondensing 6-cyano-3-hydroxypthalide (66), obtained from3-cyanophthalide (64), with phthalide (67). The product was reacted withvarious substituted aminopropyl compounds to yield indenoisoquinolines72-74 (Scheme B7).

Compound 66 was also condensed with 5,6-methylenedioxyphthalide (75,Scheme B8) to provide the methylenedioxy-substituted compound 76, whichwas reacted N′,N′-dimethylaminopropylamine (71) to yieldindenoisoquinoline 77.

Compound 85 (Scheme B9) was prepared according to previous proceduresfrom 5-nitrohomophthalic acid (47) and Schiff base 78. The nitro groupof compound 80 was reduced to aniline 81 by catalytic hydrogenation. Theamine functionality of compound 81 was replaced by an iodine atom usingSandmeyer chemistry to provide indenoisoquinoline 82, which wasconverted to the amino analogue 84 by azide displacement and Staudingerreduction. Reaction with formaldehyde under Borch reduction conditionsafforded the dimethylamino analogue 85.

Finally, to expand the diversity of the set, the effect of combining ananiline group at the 3-position with a methoxy group at the 9-positionwas investigated. In order to accomplish this, compound 86 (Scheme B10)was prepared following published procedures. Reduction of the nitrogroup of analogue 86 was attempted with several conditions, includingRaney nickel as previously done. Ultimately, it was discovered thatusing comparable amounts (by weight) of 5% Pd—C and indenoisoquinolines86 while hydrogenating at 1 atm in THF provided the most consistent andreproducible yield of intermediate 87. Treatment of compound 87 withsodium azide in DMSO at 100° C. provided analogue 88, which wassubsequently reduced with triethyl phosphite to provide compound 89,isolated as the dihydrochloride salt (Scheme B10).

Biological Results and Discussion

The IC₅₀ of the oxalic acid derivative 14 vs Tdp1 was 61±7 μM as shownin the titration curve (FIG. 14). Surface plasmon resonance studiesindicated that the compound binds the protein in a 1:1 ratio (FIG. 15).The association and dissociation rates were very fast, but within thelimit of detection of the instrument. The experiment also indicated thatthe compound did not bind to the single-stranded DNA substrate. Based onprevious results and the activity of compound 14, computational studiesusing GOLD and Sybyl were performed. Docking 14 in the catalytic site ofTdp1 suggested interactions between the oxygen atoms of 14 and the Tdp1residues Thr261, His263, Thr281, Asn283 and Ser400 (FIG. 16). Thus, aseries of compounds containing homologous side chains and ester moietiesat the 3-position were prepared in order to probe the left side of thebinding pocket as it is portrayed in FIG. 16.

TABLE B1 Tdp1 and Top1 Inhibitory Activity Compd Tdp1 Top1 CompdTdp1^(a) Top1^(b) 1 ++ +++ 40 ++(+) ++(+) 2 +++ NT 41 0 0 9 0 0 42 0 010 0 0 43 +++ 0 11 0 (+) 49 0 +++ 12 0 + 50 0 + 13 0 0 51 ++ ++ 14 + ++52 (+) +++ 15 0  +(+) 53 0 ++ 18 0 0 54 +++ ++++ 19 0 0 55 +++ ++++ 20 00 56 0 ++ 22  +(+) 0 57 ++ 0 23 ++(+) ++(+) 58 0 + 24 + (+) 59 +  +(+)25 + 0 60  +(+) ++(+) 26 + (+) 61 0 0 29 + (+) 62 ++(+) ++(+) 30 0 0 630 ++ 31 + (+) 72 0 ++ 32 0 + 73 0 ++ 33 0 + 74 ++ +++ 34 0 + 77 0 ++(+)35 0 0 84 +++  +(+) 36 0 + 85  +(+) +++ 38 0 0 89 +++ ++(+) ^(a)Tdp1IC₅₀ was determined by duplicate using a semiquantitative scale: 0,IC₅₀ >111 μM; +, IC₅₀ between 37-111 μM; ++, IC₅₀ between 12-37 μM; +++,IC₅₀ between 1-12 μM, ++++, IC₅₀ <1 μM. Active compounds were furtherevaluated for a more accurate value. ^(b)Compound-induced DNA cleavagedue to Top1 inhibition is graded by the following semiquantitative scalerelative to 1 μM camptothecin (90) or MJ-III-65 (91): 0, no detectableactivity; +, weak activity; ++, similar activity to compound 91; +++,greater activity than 91; ++++, equipotent to 90. The (+) rankingindicates the activity lies between two given values. NT: not tested.

TABLE B2 IC₅₀ Values of Tdp1 Active Compounds Compd Tdp1 (μM) 14 61 ± 722  45 ± 10 23 18 ± 8 40 11 ± 5 43  5.0 ± 1.4 51 15 ± 3 54 11 ± 1 55 5.8 ± 0.8 57 18 ± 1 62 12 ± 4 84  5.2 ± 0.1 89  6.7 ± 0.8 The IC₅₀values of Tdp1 active compounds were determined by quadruplicate

An analysis of the biological data for the rest of theindenoisoquinolines allowed the determination of some of the importantfeatures needed for Tdp1 inhibitory activity. The aminopropyl side chainis clearly important for enzyme inhibition as observed in 22, +(+); 23,++(+); 40, ++(+); 43, +++; 51, ++; 54, +++; 55, +++; 57, ++; 60, +(+);62, ++(+); 74, ++; 84, +++; 85, +(+); and 89, +++; (note: the “+ system”is a semiquantitative scale expressing IC₅₀ values at a given range asexplained in Table B1, and the IC₅₀ values of the more active Tdp1inhibitors are reported in Table B2). The indenoisoquinoline 40 waschosen as a representative example of an N-(3-aminopropyl) compound forGOLD docking and molecular mechanics energy minimization studies, whichresulted in the hypothetical binding mode displayed in FIG. 17. Thestructure suggests the existence of bonding interactions between thecharged ammonium cation of the ligand and the Val401 backbone carbonyl,the Pro461 backbone carbonyl, and the Thr466 side chain oxygen withdistances of 2.7 Å, 2.6 Å and 3.0 Å, respectively. These bondinginteractions would help to explain the Tdp1 inhibitory activity of the3-aminopropyl-substituted compounds 40 and 43 vs the inactivity of theirN-methylated and Boc-protected counterparts 38, 41, and 42.Additionally, Thr261 with its side chain hydroxyl was within H-bondingdistance, 2.7 Å, to the carbonyl oxygen of the ligand 40.

In general, compounds in which the aminopropyl side chain wasBoc-protected were either inactive or had very low Tdp1 inhibitoryactivity (Table B1). The weakly active Boc-protected compounds were 29and 31. One N-methylated compound 14 showed weak activity, +, but all ofthe other analogues containing an N-methyl side chain (9-13, 15, 38, and41) were inactive. Molecules 15, 30, and 32-36 with an acidic side chainat C-3 on the A ring were inactive but compounds 14 (+) and 43 (+++)were the exceptions. The ester-substituted indenoisoquinolinescontaining an aminopropyl side chain were all active but the activitydecreased when more methylene units were added to the ester side chain,going from +(+) and ++(+) for 22 and 23 to + for 24-26 (Table B1). Theethyl ester 40, which has a short ester side chain, was active.

Given the apparent importance of the aminopropyl side chain for Tdp1inhibition, ten compounds 54-63, see above, from our existing Top1inhibitor library were selected for testing in order to further explorethis feature. These compounds were all active as long as the3-aminopropyl side chain and the 11-keto functional group were bothpresent. Replacement of the primary amine with bromide, azide,morpholine or imidazole rendered the compounds very weak or inactive, asexemplified by 49, 50, 52, and 53 (Table B1). The primary alcohols 58and 63 were also inactive, indicating that a primary ammonium ion may bea critical feature that is important for activity as opposed to hydrogenbonding capabilities per se. Compounds 59 and 62, with hydrogen bondacceptors at the 3-position of the A ring, are also Tdp1 inhibitors. Itseems that the Tdp1 inhibitory activity of the compounds decreases asthe electron withdrawing strength of the substituent at the 3 positionincreases, as seen with the IC₅₀'s of compounds 60>57>62, which are >37,18 and 12 μM, respectively. Compounds 72-74 support the trend that anitrogen that is more basic than morpholine or imidazole on theaminopropyl side chain is necessary for Tdp1 inhibitory activity. Theprimary amine 84, +++, was more active than the tertiary amine 85, +(+),suggesting a steric limitation to binding that may also be operating inthe 72-74 series.

Molecular modeling indicates that the carbonyl of the five-membered Cring or the amide-carbonyl of the B ring may interact with either theSer400 or Thr261 side chain hydroxyl groups as seen in FIG. 16 and FIG.17. In the case of 14, the lactam carbonyl binds Thr261, whereas in 40,the ketone binds, so the ring systems are “flipped” relative to eachother. This suggests that the amino group of the ligand 40 and otherN-(3-aminopropyl)-indenoisoquinolines can play a major role in orientingthe ligand in the binding site of the enzyme.

Compound 61, which contains a hydroxyl group instead of the ketone, wasnot active, suggesting that a hydrogen bond-accepting carbonyl oxygen isimportant for activity. The presence of a 9-methoxy group on the D ringof the indenoisoquinolines seems to have a positive effect on the Tdp1inhibitory activity, as observed for 54 (+++) vs 60+(+) or 89 (+++) vs62++(+). Also, the position of the methoxy group at C7, C8, or C9affects the IC₅₀'s as seen with 51 (15 μM), 54 (11 μM) and 55 (5.8 FIG.18 shows a representative gel electrophoresis assay and titration curvesfor determination of Tdp1 IC₅₀ values for compounds 54 and 55. The9-methoxy substituent seems to play a bigger role in Tdp1 inhibitoryactivity than the substituent at the 3-position. Within the 9-methoxyindenoisoquinolines, the electron-withdrawing 3-nitro group decreasesthe activity slightly when compared with its 3-iodo or 3-aminocounterparts. The IC₅₀'s of the active compounds 54 (3-NO₂), 84 (3-I),and 89 (3-NH₂) are 11±1, 5.2±0.1, 6.7±0.8 (Table B2), respectively.Compound 77, which contains an 8,9-methylenedioxy group, was inactive vsTdp1.

The Top1 inhibitory activities of some of the active compounds havepreviously been analyzed and published. Regarding the new compounds,49-53 were less active compared with 54 and 55. For example, compounds52 and 53 present Top1 inhibitory activities of +++ and ++,respectively, compared to ++++ observed for both 54 and 55. The3-cyano-substituted compounds 72-74 showed Top1 inhibitory activities of++, ++, and +++, respectively. The 3-cyano compound 77 had good Top1activity of ++(+).

The iodo-substituted compounds 84 and 85 were also active as Top1inhibitors, although not as active as the previously published analoguessuch as 54 having a 3-nitro substituent. However, the 3-iodo substituentcould be easily exchanged, thus expanding the alternatives forsubstitution. When the nitro group of compound 54 was replaced with anamine to obtain 89, the Top1 inhibitory activity dropped from ++++ to++. The addition of a methoxy group at the 9 position of compound 60increased Top1 inhibitory activity as seen with 54 (++++) vs 60 [++(+)].However, this trend is not observed with the aniline analogues as seenin 62 with an activity of ++(+) vs 89 with an activity of ++(+).

All of the compounds were tested for induction of Top1-DNA cleavagecomplexes that are stabilized by inhibition of the DNA religationreaction due to intercalation of the drugs between DNA base pairs (FIG.19). Camptothecin (90) and the previously synthesized lead compound 91were included for comparison. The cleavage complexes were

monitored using a ³²P 3′-end labeled, 117-bp DNA fragment that wasreacted with recombinant human Top1 in the presence of increasingconcentrations of the indenoisoquinolines while separation of the DNAfragments was carried out on a denaturing gel. The sequence preferencesfor trapping the Top1-DNA cleavage complexes by the indenoisoquinolinesare similar to each other, but the pattern is different fromcamptothecin, indicating that the indenoisoquinolines target the genomedifferently from camptothecin. However, the indenoisoquinolines 52, 77,and 85 differ from each other in their abilities to suppress DNAcleavage at high drug concentrations. As is evident from the gel, theindenoisoquinolines 52 and 77, having terminal dimethylaminosubstituents on the side chain, suppress DNA cleavage at a highconcentration of 100 μM, but compound 52, having an imidazolesubstituent at the end of the chain, does not. DNA unwinding studies onsimilar 7-azaindenoisoquinolines have shown that compounds with anN-(3-dimethylaminopropyl) substituent intercalate into free DNA at highdrug concentrations, making the DNA a poorer substrate for Top1, butcompounds with an N-(3-imidazolylpropyl) substituent do not intercalateinto free DNA; so DNA cleavage is not suppressed at high drugconcentration. Although slight suppression is evident at highconcentrations of the imidazole analogue 52, it does not resemble thecomplete suppression observed with the dimethylamino analogues 77 and85.

TABLE B3 Cytotoxicities of Selected Compounds Cytotoxicity (GI₅₀ inμM)^(a) lung colon renal breast HOP- HCT- CNS melanoma ovarian SN12prostate MDA- MGM Cpd 62 116 SF-539 UACC-62 OVCAR-3 C DU-145 MB-435(μM)^(b) 22 0.65 0.57 6.90 1.22 4.01 2.53 1.11 3.36  1.86 ± 0.38^(c) 231.56 0.54 4.03 1.69 3.22 1.46 1.57 2.33 1.86 25 0.79 0.38 1.47 1.22 1.471.02 0.54 2.05 1.09 26 2.32 0.51 5.65 2.43 3.52 1.82 0.77 4.3 2.34 400.93 0.44 8.91 2.25 3.12 1.75 1.68 3.05 1.69 43 0.65 0.33 1.79 0.72 1.60.49 1.51 1.14 0.87 ± 0.014 51 0.14 0.06 0.61 0.14 0.54 0.15 0.18 0.580.17 ± 0.017 52 <0.01 <0.01 <0.01 <0.01 0.03 <0.01 <0.01 0.03  0.02 ±0.0006 54 <0.01 <0.01 <0.01 <0.01 2.82 <0.01 — 3.31  0.02 ± 0.0008 551.15 0.72 1.45 2.34 2.29 7.08 1.07 1.62 1.41 ± 0.43  60 — <0.01 0.140.03 0.08 <0.01 0.01 0.12 0.15 ± 0.10  62 0.27 0.18 0.34 0.30 0.13 0.230.12 0.23 0.16 63 0.39 0.19 0.39 0.31 0.87 0.47 0.43 1.97 0.89 ± 0.22 74 0.26 0.05 1.12 0.19 1.72 0.19 0.24 0.62 0.30 ± 0.014 84 0.14 0.070.39 0.14 1.32 0.05 0.05 0.96 0.25 ± 0.004 89 <0.01 <0.01 0.03 0.10 0.02<0.01 <0.01 0.12 0.04 ^(a)The cytotoxicity GI₅₀ values are theconcentrations corresponding to 50% growth inhibition. The compoundswere tested at concentrations ranging up to 10 μM. ^(b)Mean graphmidpoint for growth inhibition of all human cancer cell linessuccessfully tested. ^(c)For MGM GI₅₀ values in which a standard errorappears, the GI₅₀ values for individual cell lines are the average oftwo determinations; values without standard error are from onedetermination.

In order to investigate their potential as anticancer agents, a set ofindenoisoquinolines was examined for antiproliferative activity againstthe human cancer cell lines in the National Cancer Institute screen, inwhich the activity of each compound was evaluated with approximately 55different cancer cell lines of diverse tumor origins. The GI₅₀ valuesobtained with selected cell lines, along with the mean graph midpoint(MGM) values, are summarized in Table B3, above. The MGM is based on acalculation of the average GI₅₀ for all of the cell lines tested inwhich GI₅₀ values below and above the test range (10⁻⁸ to 10⁴ molar) aretaken as the minimum (10⁻⁸ molar) and maximum (10⁻⁴ molar) drugconcentrations used in the screening test. For comparison purposes, theactivities of previously reported compounds 54-63 are included on theTable B3. Many of the new compounds display significant potenciesagainst various cell lines with GI₅₀'s in the low micromolar orsubmicromolar range. All of the compounds in Table B3 except 22, 25, 43,and 63 display some degree of inhibitory activity against both Tdp1 andTop1. The most promising new compounds are 52 and 89, with mean-graphmidpoint (MGM) GI₅₀ values of 0.02 μM and 0.04 μM, respectively.Overall, the cytotoxicities do not correlate very well with thepotencies vs the isolated enzymes. For example, compounds 23, 40, and 62all have the same++(+) potencies vs both enzymes, but their cytotoxicityMGM values range from 1.86 to 0.16 μM. Another example would be 54 (MCM0.02 μM) vs 55 (MGM 1.41 μM), both of which have +++ potency vs Tdp1 and++++ potency vs Top1. Compounds 52 (MGM 0.02 μM) and 54 (MGM 0.02 μM)provide another way of stating the case, since they both have the sameMGM value but on the basis of the Tdp1 and Top1 inhibitory potencies, 54would be expected to be more cytotoxic than 52. These differences incytotoxicities may reflect different uptake, distribution, metabolism,and excretion profiles in the cellular systems as well as off-targeteffects.

Experimental Section Methyl2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetate(9)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 100 mg,0.36 mmol) was dissolved in tetrahydrofuran (10 mL) and the reactionmixture cooled to 0° C. Methyl 2-chloro-2-oxoacetate (4, 0.1 mL, 1.09mmol) was added dropwise to the indenoisoquinoline solution and thereaction mixture was stirred for 30 min. Triethylamine (0.1 mL) wasslowly added and the reaction mixture was stirred for another 30 min,keeping the temperature at 0° C. The reaction mixture was diluted withwater (30 mL) and chloroform (20 mL) was added. The organic phase wasseparated and washed with water (30 mL) and brine (30 mL). The organicphase was concentrated and the compound purified by silica gel columnchromatography, eluting with chloroform-methanol, 50:1. The product wasobtained as a brick-red solid (53 mg, 39%): mp 287-289° C. IR (Film)3336, 1642, 1514, 1480, 1406, 1358, 1333, 1231, 1045, 815, 759 cm⁻¹; ¹HNMR (300 MHz, CDCl₃) δ 11.08 (s, 1H), 8.67 (d, J=1.9 Hz, 1H), 8.40 (d,J=8.7 Hz, 1H), 8.06 (dd, J=8.8 Hz, J=2.1 Hz, 1H), 7.82 (d, J=7.2 Hz,1H), 7.50-7.43 (m, 3H), 3.93 (s, 3H), 3.87 (s, 3H); ESIMS m/z (relintensity) 363 (MH⁺, 100); HRESIMS calcd for C₂₀H₁₄N₂O₅ 363.0910 (MH⁺).found 363.0907 (MH⁺); HPLC purity: 98.3% (MeOH—H₂O, 90:10), 95.2%(MeOH—H₂O, 85:15).

Methyl3-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate(10)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 150 mg,0.54 mmol) was dissolved in tetrahydrofuran (20 mL) and the solution wascooled to 0° C. Methyl malonyl chloride (5, 0.1 mL, 0.93 mmol) was addedand the reaction mixture was stirred for 5 min at 0° C. Triethylamine(0.2 mL) was added and the reaction mixture was stirred for 2 h at 0° C.The solvent was removed under vacuum and the residue was purified bysilica gel column chromatography, eluting with chloroform-methanol,95:5, and then chloroform-ethyl acetate-methanol, 45:45:10. Thefractions were combined, the solvent was removed under vacuum and thesolid was washed with hexane-dichloromethane, 1:1 (25 mL). The productwas obtained as a reddish-brown solid (60 mg, 29%): mp 230° C. (dec). IR(Film) 3309, 3071, 2952, 1744, 1692, 1661, 1574, 1531, 1317, 1276, 1054,1017, 901 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 10.46 (s, 1H), 8.42 (d,J=1.8 Hz, 1H), 8.31 (d, J=8.8 Hz, 1H), 7.79 (dd, J=8.8 Hz, J=2.0 Hz,1H), 7.72 (d, J=7.4 Hz, 1H) 7.45-7.37 (m, 3H), 3.85 (s, 3H), 3.66 (s,3H), 3.49 (s, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.3, 168.4, 164.6,162.5, 156.0, 137.9, 137.7, 134.6, 133.9, 131.3, 127.8, 125.8, 124.3,123.7, 122.8, 117.2, 107.0, 52.4, 43.8, 32.5; ESIMS m/z (rel intensity)775 (2MNa⁺, 100), 752 (2MH⁺, 37), 377 (MH⁺, 10); HRESIMS calcd forC₂₁H₁₆N₂O₅ 377.1137 (MH⁺). found 377.1140 (MH⁺); HPLC purity: 95.0%(75:25), 95.0% (MeOH—H₂O, 70:30).

General Procedure for the Synthesis of Indenoisoquinolines 11-13

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 160 mg,0.58 mmol) was dissolved in tetrahydrofuran (15 mL) and the reactionmixture cooled to 0° C. The desired acyl chloride (6-8, 100-125 μL, 0.81mmol) was added dropwise to the indenoisoquinoline solution and thereaction mixture was stirred for 10 min. Triethylamine (0.4 mL) wasslowly added and the reaction mixture was stirred for 1 h, keeping thetemperature at 0° C. Chloroform (40 mL) was added to the reactionmixture and the organic solution was washed with water (3×40 mL) andbrine (1×50 mL). The organic phase was concentrated and the compoundspurified by silica gel column chromatography.

Methyl4-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoate(11)

The compound was eluted with chloroform-methanol, 20:1. The product wasobtained as an orange solid (101 mg, 44%): mp 250-252° C. IR (Film)3339, 3314, 2951, 1735, 1720, 1688, 1658, 1643, 1573, 1518, 1432, 1315,1195, 1157, 1055, 891, 845, 763, 700 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ10.37 (s, 1H), 8.49 (d, J=1.9 Hz, 1H), 8.35 (d, J=8.7 Hz, 1H), 7.87 (dd,J=8.7 Hz, J=2.1 Hz, 1H), 7.78 (d, J=7.5 Hz, 1H) 7.49-7.37 (m, 3H), 3.89(s, 3H), 3.57 (s, 3H), 2.61 (m, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.9,173.8, 171.0, 163.1, 156.3, 139.0, 138.3, 135.1, 134.5, 131.8, 127.9,126.3, 124.8, 124.3, 124.1, 123.3, 117.4, 107.6, 52.3, 33.0, 31.8, 29.3;ESIMS m/z (rel intensity) 413 (MNa⁺, 100); HRESIMS calcd for C₂₂H₁₈N₂O₅413.1113 (MNa⁺). found 413.1119 (MNa⁺); HPLC purity: 98.7% (MeOH—H₂O,85:15), 98.8% (MeOH—H₂O, 95:5).

Methyl5-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoate(12)

The compound was eluted with chloroform-methanol, 30:1. The product wasobtained as a dark red solid (225 mg, 96%): mp 184-186° C. IR (Film)3335, 3119, 2945, 1730, 1689, 1650, 1580, 1525, 1431, 1316, 1274, 1194,901, 844, 757, 718 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.49(d, J=1.9 Hz, 1H), 8.35 (d, J=8.7 Hz, 1H), 7.85 (dd, J=8.7 Hz, J=2.1 Hz,1H), 7.76 (d, J=7.5 Hz, 1H) 7.45-7.32 (m, 3H), 3.88 (s, 3H), 3.56 (s,3H), 2.46 (m, 4H), 1.81 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.3,173.4, 171.2, 162.5, 155.6, 138.3, 137.7, 134.5, 133.8, 131.2, 127.3,125.8, 124.2, 123.7, 123.5, 122.7, 116.9, 107.0, 51.6, 35.6, 32.9, 32.4,20.6; ESIMS m/z (rel intensity) 405 (MH⁺, 100); HRESIMS calcd forC₂₃H₂₀N₂O₅ 405.1450 (MH⁺). found 405.1446 (MH⁺); HPLC purity: 96.0%(MeOH—H₂O, 80:20).

Methyl6-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoate(13)

The compound was eluted with chloroform-methanol, 30:1. The product wasobtained as a red solid (221 mg, 91.2%): mp 177-179° C. IR (Film) 3341,3071, 2950, 2868, 1732, 1692, 1659, 1573, 1522, 1512, 1434, 1316, 1276,1196, 1055, 902, 842, 762, 750 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ 10.20(s, 1H), 8.49 (d, J=2.0 Hz, 1H), 8.36 (d, J=8.8 Hz, 1H), 7.88 (dd, J=8.7Hz, J=2.0 Hz, 1H), 7.79 (d, J=7.5 Hz, 1H) 7.51-7.36 (m, 3H), 3.91 (s,3H), 3.55 (s, 3H), 2.31 (m, 4H), 1.56 (m, 4H); ¹³C NMR (125 MHz,DMSO-d₆) δ 190.3, 173.6, 171.6, 162.5, 155.8, 138.5, 137.7, 134.6,133.9, 131.2, 127.3, 125.9, 124.2, 123.7, 123.5, 122.8, 117.0, 107.1,51.6, 36.4, 33.4, 32.5, 24.8, 24.4; ESIMS (rel intensity) m/z 419 (MH⁺,100); HRESIMS calcd for C₂₄H₂₂N₂O₅ 419.1607 (MH⁺). found 419.1613 (MH⁺);HPLC purity: 97.0% (MeOH—H₂O, 85:15), 96.9% (MeOH—H₂O, 90:10).

(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)-carbamicAcid (14)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 201 mg,0.72 mmol) was dissolved in tetrahydrofuran (30 mL) and cooled to 0° C.Methyl oxalyl chloride (4, 0.1 mL, 1.16 mmol) and triethylamine (0.2 mL)were added dropwise and the reaction mixture was stirred for 2 h at 0°C. The reaction mixture was diluted with water (100 mL) and extractedwith chloroform (4×50 mL). The solvent was removed under vacuum and thecompound passed through a short silica gel column chromatography,eluting with chloroform-methanol, 9:1. The impure solid, compound 9, wasdissolved in a solution of sodium hydroxide (0.1 g, 2.5 mmol) in ethanol(50 mL) and water (1 mL) and the reaction mixture was stirred overnightat room temperature. The solvent was removed under vacuum and theresidue purified by silica gel column chromatography, eluting withchloroform-methanol-acetic acid, 90:9:1. Compound 14 was obtained as areddish-brown solid (103 mg, 44.7%, after 2 steps): mp 360° C. (dec). IR(Film) 3325, 2928, 1692, 1657, 1600, 1580, 1533, 1435, 1319, 1197, 903,701 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 10.9 (s, 1H), 8.70 (s, 1H), 8.35(d, J=8.2 Hz, 1H), 8.02 (s, 1H), 7.90 (br s, 1H), 7.45 (m, 3H), 3.89 (s,3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.1, 162.4, 162.2, 157.9, 156.3,137.5, 136.9, 134.6, 134.0, 131.4, 128.4, 126.9, 124.4, 123.5, 123.4,122.8, 118.5, 106.9; ESIMS m/z (rel intensity) 347 [(M-H⁺)⁻]; negativeion; HRESIMS calcd for C₁₉H₁₂N₂O₅ 347.0668 [(M-H⁺)⁻]. found 347.0664[(M-H⁺)⁻]; HPLC purity: 95.2% (MeOH—H₂O, 85:15), 95.7% (MeOH—H₂O,75:25).

3-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoicAcid (15)

Methyl3-[(6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate(10, 158 mg, 0.42 mmol) was dissolved in methanol (20 mL) anddimethylformamide (2 mL). A solution of sodium hydroxide (103 mg) inwater (2 mL) was added dropwise and the reaction mixture was stirred atroom temperature for 24 h. Concentrated hydrochloric acid (1 mL) wasadded and the solution diluted with chloroform (30 mL). A dark-redprecipitated was formed between the two phases. The solid was filteredand the organic and water layers discarded. The solid was washed withwater (10 mL) and ether (2×10 mL). The solid was dried and compound 15was obtained as a reddish-brown solid (93 mg, 61%): mp 262° C. (dec). IR(KBr) 3447, 3323, 1731, 1695, 1573, 1529, 1433, 1317, 1195, 1054, 899,763 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 10.5 (s, 1H), 8.45 (s, 1H), 8.31(d, J=8.6 Hz, 1H), 7.80 (d, J=7.0 Hz, 1H), 7.46-7.38 (m, 3H), 3.86 (s,3H), 3.39 (s, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.1, 169.5, 165.1,162.4, 155.7, 138.1, 137.5, 134.5, 133.8, 131.2, 127.6, 125.7, 124.1,123.6, 122.7, 117.0, 116.9, 106.9, 44.4, 32.4; MALDIMS m/z (relintensity) 363 (MH⁺, 100); ESIMS (m/z, relative intensity) 361[(M-H⁺)⁻], 100); HRESIMS calcd for C₂₀H₁₄N₂O₅ 361.0824 [(M-H⁺)⁻]. found361.0828 [(M-H⁺)⁻]; negative ion; HPLC purity: 99.4% (MeOH, 100), 98.6%(MeOH—H₂O, 95:5).

Methyl2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetate(17)

Compound 16 (0.3 g, 0.715 mmol) was dissolved in chloroform (100 mL).Triethylamine (0.261 g, 2.14 mmol) was added at room temperaturefollowed by methyl oxaloyl chloride (4, 0.113 g, 0.930 mmol) and thereaction mixture was stirred for 2 h. The reaction mixture was washedwith water (2×20 mL) and dried over anhydrous sodium sulfate. Thesolvent was removed under vacuum and the residue purified by silica gelcolumn chromatography, eluting with chloroform-methanol, 9.4:0.4, tofurnish the product 17 (0.230 g, 65%) as an orange solid: mp 157-159° C.IR (KBr) 3339, 2976, 1702, 1662, 1580, 1570, 1534, 1514, 1163, 759, 665cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 9.02 (s, 1H), 8.73 (d, J=8.7 Hz, 1H),8.32 (s, 1H), 8.10 (dd, J=1.8, J=5.6 Hz, 1H), 7.68 (m, 1H), 7.48 (m,1H), 7.30 (m, 2H), 5.30 (m, 1H), 4.58 (t, J=4.8 Hz, 2H), 3.99 (s, 3H),3.26 (m, 2H), 2.10 (m, 2H), 1.45 (s, 9H); ¹³C NMR (CDCl₃+CD₃OD, 75 MHz)δ 163.2, 160.6, 156.5, 154.6, 136.5, 135.6, 134.5, 133.4, 130.9, 129.2,126.4, 124.3, 123.5, 123.1, 122.5, 118.3, 108.4, 79.3, 53.6, 42.2, 37.2,29.5, 28.0; ESIMS m/z (relative intensity) 528 (MNa⁺, 62); HRESIMS calcdfor C₂₇H₂₇N₃O₇Na 528.1747 (MNa⁺). found 528.1740 (MNa⁺).

Methyl3-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate(18)

Compound 16 (0.100 g, 0.234 mmol) was dissolved in chloroform (75 mL),triethylamine (0.060 g, 0.591 mmol) was added followed by methyl3-chloro-3-oxopropionate (5, 0.055 g, 0.290 mmol) at room temperature,and the reaction mixture was stirred for 2 h. The reaction mixture waswashed with water (2×20 mL), extracted with chloroform (2×75 mL), anddried over anhydrous sodium sulfate. The solvent was removed undervacuum and the residue purified by silica gel column chromatography,eluting with chloroform-methanol, 9.3:0.7, to furnish the product 18(0.075 g, 65%) as a red solid: mp 144-145° C. IR (KBr) 3325, 2976, 1744,1696, 1664, 1572, 1532, 1512, 1433, 1168, 761, 665 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 9.47 (s, 1H), 8.65 (d, J=8.8 Hz, 1H), 8.35 (s, 1H), 8.06 (m,1H), 7.59 (m, 1H), 7.50 (m, 1H), 7.41 (m, 2H), 5.40 (br s, 1H), 4.60 (t,J=6.6 Hz, 2H), 3.82 (s, 3H), 3.53 (s, 2H), 3.50 (m, 2H), 2.08 (m, 2H),1.45 (s, 9H); ¹³C NMR (CDCl₃, 300 MHz) δ 190.0, 169.8, 163.3, 163.0,156.1, 154.1, 136.7, 136.5, 134.6, 133.2, 130.6, 128.5, 126.5, 124.2,123.6, 123.0, 122.3, 118.2, 108.3, 79.2, 52.6, 42.0, 41.7, 37.3, 29.9,28.4; ESIMS m/z (relative intensity) 542 (MNa⁺, 100); HRESIMS calcd forC₂₈H₂₉N₃O₇Na 542.1903 (MNa⁺). found 542.1906 (MNa⁺); 95.7% (MeOH—H₂O,85:15).

Methyl4-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoate(19)

Compound 16 (0.150 g, 0.357 mmol) was dissolved in chloroform (100 mL),triethylamine (0.090 g, 0.892 mmol) was added followed by methyl4-chloro-4-oxobutanoate (6, 0.082 g, 0.536 mmol) at room temperature,and the reaction mixture was stirred for 2 h. The reaction mixture waswashed with water (2×20 mL), extracted with chloroform (2×75 mL), anddried over anhydrous sodium sulfate. The solvent was removed undervacuum and the residue purified by silica gel column chromatography,eluting with chloroform-methanol, 9.5:0.5, to furnish the product 19(0.114 g, 60%) as an orange solid: mp 176-178° C. IR (KBr) 3307, 2917,1728, 1703, 1718, 1695, 1575, 1165, 666 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ8.68 (d, J=5.3 Hz, 1H), 8.21 (br s, 1H), 8.02 (m, 2H), 7.68 (d, J=4.8Hz, 1H), 7.32 (m, 4H), 5.41 (br s, 1H), 4.57 (t, J=3.2 Hz, 2H), 3.72 (s,3H), 3.20 (m, 2H), 2.79 (m, 4H), 2.08 (m, 2H), 1.45 (s, 9H); ¹³C NMR(CDCl₃+DMSO-d₆, 125 MHz) δ 190.0, 173.0, 170.0, 162.9, 155.8, 153.6,137.9, 136.7, 134.5, 133.0, 130.3, 127.4, 126.0, 123.7, 123.4, 122.6,122.0, 117.2, 108.3, 78.7, 51.4, 41.6, 36.9, 31.1, 29.6, 28.6, 28.1;ESIMS m/z (relative intensity) 556 (MNa⁺, 12); HRESIMS calcd forC₂₉H₃₁N₃O₇Na 556.2060. found 556.2069; HPLC purity: 95.0% (MeOH—H₂O,85:15).

Methyl5-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoate(20)

Compound 16 (0.150 g, 0.357 mmol) was dissolved in chloroform (100 mL),triethylamine (0.108 g, 1.07 mmol) was added followed by methyl5-chloro-5-oxopentanoate (7, 0.088 g, 0.536 mmol) at room temperature,and the reaction mixture was stirred for 2 h. The reaction mixture waswashed with water (2×25 mL), extracted with chloroform (2×60 mL), anddried over anhydrous sodium sulfate. The solvent was removed undervacuum and the residue purified by silica gel column chromatography,eluting with chloroform-methanol, 9.6:0.4, to afford the product 20 (0.1g, 50%) as a red solid: mp 118-120° C. IR (KBr) 3329, 1696, 1679, 1663,1596, 1528, 1169, 760 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.52 (d, J=5.3 Hz,1H), 8.21 (br s, 1H), 8.01 (br s, 1H), 7.48 (d, J=4.7 Hz, 1H), 7.40 (m,2H), 7.26 (m, 1H), 5.48 (t, J=4.5 Hz, 1H), 4.51 (t, J=4.5 Hz, 2H), 3.69(s, 3H), 3.20 (m, 2H), 2.49 (m, 4H), 2.10 (m, 4H), 1.45 (s, 9H); ¹³C NMR(CDCl₃, 125 MHz) δ 190.2, 173.8, 170.8, 163.2, 156.1, 154.0, 137.1,136.9, 134.7, 133.3, 130.7, 128.3, 126.6, 124.3, 123.7, 123.1, 122.3,117.9, 108.6, 79.3, 51.7, 41.9, 37.3, 36.1, 32.9, 30.0, 28.4, 20.6;ESIMS m/z (relative intensity) 570 (MNa⁺, 100); HRESIMS calcd forC₃₀H₃₃N₃O₇Na 570.2216 (MNa⁺). found 570.2209 (MNa⁺); HPLC purity: 96.4%(MeOH—H₂O, 85:15).

Methyl6-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoate(21)

Compound 16 (0.150 g, 0.357 mmol) was dissolved in chloroform (100 mL),triethylamine (0.090 g, 0.892 mmol) was added followed by methyl6-chloro-6-oxohexanoate (8, 0.095 g, 0.536 mmol) at room temperature,and the reaction mixture was stirred for 2 h. The reaction mixturewashed with water (2×30 mL), extracted with chloroform (2×75 mL), anddried over anhydrous sodium sulfate. The solvent was removed undervacuum and the residue purified by silica gel column chromatography,eluting with chloroform-methanol, 9.7:0.3, to yield the product 21(0.116 g, 55%) as a red solid: mp 143-145° C. IR (KBr) 3332, 2974, 1696,1662, 1580, 1568, 1527, 1169, 760, 666 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ8.46 (d, J=8.7 Hz, 1H), 8.28 (br s, 1H), 8.03 (br s, 1H), 7.93 (d, J=5.2Hz, 1H), 7.52 (d, J=5.2 Hz, 1H), 7.31 (m, 2H), 7.28 (d, J=6.3 Hz, 1H),5.43 (t, J=5.4 Hz, 1H), 4.48 (t, J=6.6 Hz, 2H), 3.67 (s, 3H), 3.18 (m,2H), 2.38 (m, 4H), 2.05 (m, 2H), 1.76 (m, 4H), 1.45 (s, 9H); ¹³C NMR(CDCl₃, 125 MHz) δ 190.3, 174.1, 171.1, 163.1, 156.1, 154.1, 137.2,136.9, 134.8, 133.3, 130.7, 128.3, 126.7, 124.3, 123.7, 123.1, 122.3,118.0, 108.6, 79.3, 51.6, 41.9, 37.3, 37.0, 33.6, 30.0, 28.4, 24.8,24.2; ESIMS m/z (relative intensity) 584 (MNa⁺, 17); HRESIMS calcd forC₃₁H₃₅N₃O₇Na 584.2373. found 584.2370.

Methyl2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoacetate(22)

Compound 17 (0.070 g, 0.130 mmol) was treated with trifluoroacetic acid(0.5 mL) in chloroform (5 mL) for 2 h at room temperature. The solventwas removed on a rotary evaporator and the residue was then basifiedwith 2 N NH₃ in methanol to get the free amine, which was purified bysilica gel column chromatography, eluting with chloroform-methanol,8.7:1.3, to yield the product 22 (0.035 g, 65%) as a brown solid: mp185-186° C. IR (KBr) 3014, 1692, 1581, 1570, 1533, 1307, 1198, 722, 455cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.55 (s, 1H), 8.23 (d, J=3.2 Hz, 1H),7.71 (d, J=3.5 Hz, 1H), 7.54 (m, 1H), 7.36 (m, 3H), 4.52 (m, 2H), 3.91(s, 3H), 3.05 (m, 2H), 2.28 (m, 2H); ¹³C NMR (CD₃OD, 125 MHz) δ 191.6,169.9, 166.8, 164.9, 155.4, 138.8, 137.9, 135.7, 134.9, 132.2, 129.6,127.2, 125.0, 124.8, 124.1, 123.9, 118.6, 109.6, 52.9, 42.5, 38.2, 28.6;ESIMS m/z (relative intensity) 420 (MH⁺, 100); HRESIMS calcd forC₂₃H₂₁N₃O₅ 420.1559 (MH⁺). found 420.1554 (MH⁺); HPLC purity: 98.2% (1%TFA in MeOH).

Methyl3-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoate(23)

Compound 18 (0.080 g, 0.145 mmol) was treated with TFA (0.5 mL) inchloroform (5 mL) for 2 h at room temperature. The solvent was removedon a rotary evaporator and the residue was then basified with 2 N NH₃ inmethanol to get the free amine, which was purified by silica gel columnchromatography, eluting with chloroform-methanol, 8.8:1.2, to yield theproduct 23 (0.040 g, 62%) as a brown solid: mp 225-227° C. IR (KBr)3067, 1739, 1673, 1574, 1535, 1511, 1202, 1134, 722 cm⁻¹; ¹H NMR (CD₃OD,300 MHz) 8.52 (d, J=2.1 Hz, 1H), 8.29 (d, J=6.8 Hz, 1H), 7.43 (m, 5H),4.50 (t, J=2.1 Hz, 2H), 3.78 (s, 3H), 3.10 (m, 2H), 2.22 (m, 2H); ¹³CNMR (CD₃OD, 125 MHz) δ 191.6, 169.9, 166.8, 164.9, 155.4, 138.8, 137.9,135.7, 134.9, 132.2, 129.6, 127.2, 125.0, 124.8, 124.1, 123.9, 118.6,109.6, 52.9, 42.5, 38.2, 28.6; ESIMS m/z (relative intensity) 420 (MH⁺,100); HRESIMS calcd for C₂₃H₂₁N₃O₅ 420.1559 (MH⁺). found 420.1554 (MH⁺);HPLC purity: 100% (1% TFA in MeOH—H₂O, 50:50).

Methyl4-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoate(24)

Compound 19 (0.060 g, 0.112 mmol) was treated with trifluoroacetic acid(0.5 mL) in chloroform (5 mL) for 2 h at room temperature. The solventwas removed on a rotary evaporator and the residue was then basifiedwith 2 N NH₃ in in methanol to get the free amine, which was purified bysilica gel column chromatography, eluting with chloroform-methanol,8.8:1.2, to yield the product 24 (0.028 g, 60%) as a brown solid: mp272-274° C. IR (KBr) 2952, 1735, 1690, 1656, 1572, 1532, 1510, 1160,765, 455 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.28 (s, 1H), 7.92 (m, 1H),7.31 (m, 3H), 7.20 (s, 2H), 4.30 (t, J=4.8 Hz, 2H), 3.71 (s, 3H), 2.83(m, 2H), 2.68 (m, 4H), 2.00 (m, 2H); ¹³C NMR (CD₃OD, 125 MHz) δ 191.8,174.9, 172.6, 164.4, 155.6, 139.2, 138.2, 135.9, 134.8, 131.9, 129.3,127.1, 125.0, 124.9, 124.0, 123.8, 122.9, 118.5, 114.8, 109.5, 57.6,57.4, 57.2, 43.6, 36.3, 32.2, 30.3, 29.7; ESIMS m/z (relative intensity)434 (MH⁺, 100); HRESIMS calcd for C₂₄H₂₃N₃O₅ 434.1852 (MH⁺). found434.1835 (MH⁺); HPLC purity: 98.0% (1% TFA in MeOH).

Methyl5-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoate(25)

Compound 20 (0.150 g, 0.274 mmol) was treated with trifluoroacetic acid(1.0 mL) in chloroform (10 mL) for 2 h at room temperature. The solventwas removed on a rotary evaporator and the residue was then basifiedwith 2 N NH₃ in methanol to get the free amine, which was purified bysilica gel column chromatography, eluting with chloroform-methanol,9.0:1.0, to afford the product 25 (0.092 g, 75%) as a brown solid: mp215-217° C. IR (KBr) 3075, 1729, 1687, 1673, 1572, 1532, 1433, 1202,760, 722, 455 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 10.2 (s, 1H), 8.60 (d,J=1.8 Hz, 1H), 8.47 (d, J=8.7 Hz, 1H), 7.89 (dd, J=1.8, J=8.7 Hz, 1H),7.74 (m, 3H), 7.54 (m, 2H), 7.47 (m, 1H), 4.53 (t, J=3.2 Hz, 2H), 3.59(s, 3H), 2.96 (m, 2H), 2.38 (m, 4H), 2.13 (m, 2H), 1.87 (m, 2H); ¹³C NMR(DMSO-d₆, 75 MHz) δ 190.1, 173.0, 171.0, 162.6, 154.4, 138.3, 136.6,134.2, 134.0, 131.0, 127.1, 125.8, 123.5, 123.4, 122.6, 116.6, 107.4,79.1, 51.3, 41.2, 38.6, 36.6, 35.2, 32.6, 27.3, 20.2; ESIMS m/z(relative intensity) 448 (MH⁺, 100); HRESIMS calcd for C₂₅H₂₅N₃O₅448.1867 (MH⁺). found 448.1877 (MH⁺); HPLC purity: 100% (1% TFA inMeOH—H₂O, 70:30).

Methyl6-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoate(26)

Compound 21 (0.030 g, 0.053 mmol) was treated with trifluoroacetic acid(0.25 mL) in chloroform (3 mL) for 2 h at room temperature. The solventwas removed on a rotary evaporator and the residue was then basifiedwith 2 N NH₃ in methanol to get the free amine, which was purified bysilica gel column chromatography, eluting with chloroform-methanol,9.2:0.8, to yield the product 26 (0.015 g, 55%) as a red solid: mp153-155° C. IR (KBr) 2947, 1739, 1705, 1693, 1661, 1570, 1530, 1431,1197, 760, 665 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.51 (d, J=2.4 Hz, 1H),8.34 (d, J=9.0 Hz, 1H), 7.70 (dd, J=2.4, J=9.0 Hz, 1H), 7.60 (t, J=9.0Hz, 1H), 7.51 (m, 2H), 7.31 (m, 1H), 4.55 (t, J=4.5 Hz, 2H), 3.67 (s,3H), 3.13 (m, 2H), 2.39 (m, 4H), 2.26 (m, 2H), 1.70 (m, 2H); ¹³C NMR(DMSO-d₆, 125 MHz) δ 190.0, 173.3, 171.3, 162.3, 154.4, 138.3, 136.6,134.2, 133.9, 130.9, 127.0, 125.6, 123.5, 123.4, 123.3, 122.5, 116.5,107.2, 51.2, 42.1, 38.2, 36.0, 33.0, 30.9, 24.4, 24.0; ESIMS (m/z,relative intensity) 462 (MH⁺, 100); HRESIMS calcd for C₂₆H₂₇N₃O₅462.2023 (MH⁺). found 462.2031 (MH⁺); HPLC purity: 98.5% (1% TFA inMeOH—H₂O, 70:30).

General Procedure for Synthesis of Indenoisoquinolines 27-31

The esters 17-21 (0.1 g) were dissolved in methanol (10 mL) andtetrahydrofuran (10 mL). An aqueous NaOH solution (4 N, 5 mL) was addedat room temperature and the reaction mixture was stirred at roomtemperature for 6 h. The solvent was removed on a rotary evaporator andthe residue washed with 1 N HCl (30 mL), extracted with chloroform (2×50mL), dried over anhydrous sodium sulfate, concentrated and purified bysilica gel column chromatography, eluting with chloroform-methanol9.6:0.4 to 9:1, to afford acids 27-31 in 55-75% yields as orange solids.

2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoaceticAcid (27)

mp 267-268° C. IR (KBr) 3295, 1758, 1698, 1646, 1605, 1580, 1535, 1350,1161, 847, 665 cm⁻¹; ¹H NMR (CD₃OD+DMSO-d₆, 300 MHz) δ 8.87 (s, 1H),8.63 (d, J=8.7 Hz, 1H), 8.14 (d, J=9.3 Hz, 1H), 7.80 (d, J=7.2 Hz, 1H),7.64 (m, 2H), 7.55 (m, 1H), 4.60 (t, J=7.2 Hz, 2H), 3.21 (m, 2H), 2.07(m, 2H), 1.50 (s, 1H).

3-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoicAcid (28)

mp 211-212° C. IR (KBr) 3310, 1768, 1710, 1976, 1522, 1385, 1355, 847,665 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.56 (d, J=2.2 Hz, 1H), 8.30 (d,J=5.6 Hz, 1H), 7.51 (m, 1H), 7.48 (m, 1H), 7.44 (m, 1H), 7.39 (m, 1H),7.30 (m, 1H), 4.52 (t, J=4.5 Hz, 2H), 3.52 (s, 2H), 3.15 (t, J=5.8 Hz,2H), 2.18 (m, 2H), 1.43 (s, 9H).

4-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoicAcid (29)

mp 155-157° C. IR (KBr) 3315, 1763, 1698, 1678, 1543, 1345, 1165, 667cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.44 (m, 211), 7.82 (dd, J=2.1, J=5.5Hz, 1H), 7.68 (d, J=5.5 Hz, 1H), 7.51 (m, 2H), 7.34 (m, 1H), 4.45 (m,2H), 3.12 (m, 2H), 2.14 (s, 4H), 1.96 (m, 2H), 1.44 (s, 9H); ¹³C NMR(CDCl₃+DMSO-d₆, 75 MHz) δ 188.4, 172.3, 168.8, 160.8, 154.3, 152.3,136.8, 135.3, 133.0, 131.8, 129.0, 125.7, 124.0, 122.1, 121.9, 121.2,120.9, 115.2, 106.2, 40.9, 35.9, 29.7, 29.1, 28.1, 27.3, 26.7; ESIMS(m/z, relative intensity) 542 (MNa⁺, 100), 420 (loss of Boc, 16);HRESIMS calcd for C₂₈H₂₉N₃O₇Na 542.1903 (MNa⁺). found 542.1912 (MNa⁺).

5-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoicAcid (30)

mp 198-200° C. IR (KBr) 3307, 1696, 1690, 1663, 1569, 1528, 1401, 1366,1250, 1167, 759 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.52 (d, J=5.3 Hz, 1H),8.21 (br s, 1H), 8.01 (br, s, 1H), 7.48 (d, J=4.7 Hz, 1H), 7.40 (m, 2H),7.26 (m, 1H), 4.51 (t, J=4.5 Hz, 2H), 3.20 (m, 2H), 2.49 (m, 4H), 2.10(m, 4H), 1.45 (s, 9H); ¹³C NMR (CD₃OD+DMSO-d₆, 75 Hz) δ 191.5, 176.3,173.2, 164.1, 158.0, 155.6, 139.3, 138.2, 135.9, 134.8, 131.9, 129.0,127.0, 125.0, 124.8, 124.2, 123.8, 118.4, 109.1, 79.8, 79.7, 36.9, 34.2,30.9, 29.0, 21.9; ESIMS (m/z, relative intensity) 556 (MNa⁺, 100);HRESIMS calcd for C₂₉H₃₁N₃O₇ 556.2059 (MNa⁺). found 556.2063 (MNa⁺).

6-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoicAcid (31)

mp 206-208° C. IR (KBr) 3312, 1702, 1695, 1670, 1573, 1534, 1376, 1250,760, 665 cm⁻¹; ¹H NMR (CDCl₃+CD₃OD, 300 MHz) δ 8.55 (d, J=6.2 Hz, 1H),8.18 (s, 1H), 8.01 (m, 1H), 7.50 (d, J=3.5 Hz, 1H), 7.40 (d, J=3.5 Hz,1H), 7.32 (m, 1H), 7.28 (d, J=3.5 Hz, 1H), 4.50 (m, 2H), 3.12 (m, 2H),2.29 (m, 4H), 1.96 (m, 2H), 1.65 (m, 4H), 1.37 (s, 9H); ¹³C NMR(DMSO-d₆, 75 Hz) δ 190.0, 174.5, 171.5, 162.2, 155.8, 154.5, 138.3,136.7, 134.3, 133.9, 131.0, 127.1, 125.7, 123.5, 123.3, 123.2, 122.7,116.6, 107.3, 77.8, 42.5, 37.5, 36.2, 33.5, 29.6, 28.3, 24.6, 24.9;ESIMS (m/z, relative intensity) 570 (MNa⁺, 23), 448 (loss of Boc, 100);HRESIMS calcd for C₃₀H₃₃N₃O₇Na 570.2216 (MNa⁺). found 570.2207 (MNa⁺).

General Procedure for Synthesis of Indenoisoquinolines 32-36

Acids 27-31 (0.050 g) were treated with HCl in diethyl ether (2 M, 2 mL)at room temperature for 5 h. The solvent was removed on a rotaryevaporator to yield the solid hydrochloride salts, which were washedwith 5% MeOH in chloroform to remove impurities and afford the pureproducts 32-36 in quantitative yields.

2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-2-oxoaceticAcid Hydrochloride (32)

mp 251-252° C. IR (KBr) 2955, 1742, 1733, 1641, 1578, 1535, 1431, 1195,665 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 11.04 (s, 1H), 8.75 (dd, J=1.7,J=8.9 Hz, 1H), 8.49 (dd, J=4.0, J=8.8 Hz), 8.11 (m, 2H), 7.69 (m, 1H),7.54 (m, 3H), 4.54 (m, 2H), 3.01 (m, 2H), 2.07 (m, 2H); ESIMS (m/z,relative intensity) 392 (MH⁺, 100); HRESIMS calcd for C₂₁H₁₇N₃O₅392.1246 (MH⁺). found 392.1249 (MH⁺); HPLC purity: 96.6% (1% TFA inMeOH).

3-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-3-oxopropanoicAcid Hydrochloride (33)

mp 205-207° C. IR (KBr) 2917, 2356, 1670, 1582, 1565, 1538, 1191, 665cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 10.65 (d, J=8.9 Hz, 1H), 8.48 (s, 1H),8.40 (d, J=4.5 Hz, 1H), 7.88 (m, 4H), 7.71 (d, J=4.5 Hz, 1H), 7.56 (m,2H), 7.41 (m, 1H), 4.54 (m, 2H), 3.64 (s, 1H), 3.52 (s, 1H), 3.00 (m,2H), 2.09 (m, 2H); ESIMS (m/z, relative intensity) 406 (MH⁺, 100);HRESIMS calcd for C₂₂H₁₉N₃O₅ 406.1403 (MH⁺). found 406.1400 (MH⁺); HPLCpurity: 95.2% (1% TFA in MeOH).

4-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-4-oxobutanoicAcid Hydrochloride (34)

mp 167-169° C. IR (KBr) 3080, 1671, 1571, 1533, 1508, 1197, 734, 665cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 8.68 (d, J=2.3 Hz, 1H), 8.55 (d, J=5.2Hz, 1H), 7.78 (dd, J=2.3, J=5.4 Hz, 1H), 7.64 (m, 1H), 7.52 (m, 2H),7.37 (m, 1H), 4.62 (m, 2H), 3.06 (m, 2H), 2.30 (m, 2H); ESIMS (m/z,relative intensity) 420 (MH⁺, 100); HRESIMS calcd for C₂₃H₂₁N₃O₅420.1559 (MH⁺). found 420.1565 (MH⁺; 96.71% (1% TFA in MeOH).

5-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-5-oxopentanoicAcid Hydrochloride (35)

mp 242-244° C. IR (KBr) 3583, 2348, 1761, 1723, 1695, 1510, 1497, 906,673 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 10.33 (s, 1H), 8.63 (d, J=2.1 Hz,1H), 8.49 (d, J=8.7 Hz, 1H), 7.89 (dd, J=2.1, J=8.7 Hz, 1H), 7.84 (br s,2H), 7.76 (d, J=7.2 Hz, 1H), 7.54 (m, 2H), 7.40 (m, 1H), 4.53 (m, 2H),3.04 (m, 2H), 2.40 (m, 2H), 2.04 (m, 4H), 2.13 (m, 2H), 1.85 (m, 2H);ESIMS (m/z, relative intensity) 434 (MH⁺, 100); HRESIMS calcd forC₂₄H₂₃N₃O₅ 434.1716. found 434.1710; HPLC purity: 95.3% (1% TFA inMeOH).

6-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]-6-oxohexanoicAcid Hydrochloride (36)

mp 203-205° C. IR (KBr) 3583, 2952, 1730, 1719, 1646, 1570, 1528, 1431,1195, 759, 667 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 10.30 (d, J=6.6 Hz,1H), 8.85 (d, J=9.9 Hz, 1H), 8.51 (m, 1H), 7.91 (m, 1H), 7.76 (m, 3H),7.51 (m, 3H), 4.50 (m, 2H), 3.32 (m, 1H), 3.22 (m, 1H), 2.39 (m, 2H),2.27 (m, 2H), 2.11 (m, 1H), 1.91 (m, 1H), 1.58 (m, 4H); ESIMS (m/z,relative intensity) 448 (MH⁺, 100); HRESIMS calcd for C₂₅H₂₆N₃O₅448.1872 (MH⁺). found 448.1868 (MH⁺); HPLC purity: 96.5% (1% TFA inMeOH).

Ethyl2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]acetate(38)

3-Amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (3, 400 mg,1.45 mmol) and ethyl glyoxylate (37, 0.3 mL, 50% solution in toluene)were dissolved in glacial acetic acid (25 mL). The reaction mixture wasstirred overnight at room temperature. Sodium cyanoborohydride (1.00 g)was added in portions. Once the production of bubbles stopped, thereaction mixture was heated at reflux for 30 min. Water (150 mL) wasadded and the compound extracted with chloroform (3×50 mL). The organiclayers were combined and washed with water (2×150 mL), aqueous sodiumbicarbonate (100 mL) and brine (100 mL). The solvent was removed undervacuum and the compound purified by silica gel column chromatography,eluting with chloroform-methanol, 50:1. The product was obtained as adark solid (351 mg, 67%): mp 207° C. (dec). IR (Film) 3380, 2929, 1735,1694, 1654, 1560, 1434, 1212, 1017 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆) δ8.21 (d, J=8.7 Hz, 1H), 7.69 (d, J=7.5 Hz, 1H), 7.43-7.38 (m, 2H), 7.31(t, J=7.4 Hz, 1H), 7.13 (dd, J=8.8 Hz, J=2.5 Hz, 1H), 7.10 (d, J=2.5 Hz,1H), 6.67 (t, J=6.4 Hz, 1H), 4.09 (q, J=7.1 Hz, 2H), 3.96 (d, J=6.3 Hz,2H), 3.87 (s, 3H), 1.12 (t, J=7.1 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ190.7, 170.6, 163.0, 152.2, 146.1, 138.3, 134.8, 132.8, 130.0, 125.0,124.7, 123.7, 122.9, 121.9, 121.2, 109.0, 107.0, 61.4, 45.3, 32.2, 14.1;ESIMS m/z (rel intensity) 363 (MH⁺, 100); HRESIMS calcd for C₂₁H₁₈N₂O₄363.1345 (MH⁺). found 363.1349 (MH⁺); HPLC purity: 99.42% (MeOH—H₂O-TFA,95:5:1), 95.33% (MeOH-TFA, 100:1).

Ethyl2-[(6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl]amino)acetate(39)

Compound 16 (0.200 g, 0.477 mmol) was dissolved in acetic acid (20 mL)and methanol (1 mL). Ethyl glyoxalate (37, 0.058 g, 0.577 mmol) wasadded to the reaction mixture, which was stirred at room temperature for1.5 h. Sodium cyanoborohydride (0.071 g, 1.19 mmol) was added andstirring was continued for another 0.5 h. The solvents were removed on arotary evaporator. The residue was washed with sodium bicarbonate (2×15mL), extracted with CHCl₃ (2×60 mL), the combined organic layers weredried over anhydrous sodium bicarbonate, and the mixture wasconcentrated to get a crude product. The crude product was precipitatedfrom hexane-chloroform (7+3 mL) to afford a pure violet solid 39 (0.215g, 90%): mp 203-204° C. IR (KBr) 2977, 1739, 1698, 1650, 1619, 1579,1523, 1390, 1365, 1198, 1171, 1020, 758, 455 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 8.51 (d, J=8.7 Hz, 1H), 7.53 (d, J=6.4 Hz), 7.39 (m, 4H), 7.09(dd, J=2.6, J=8.7 Hz, 1H), 5.44 (m, 1H), 4.66 (m, 1H), 4.56 (t, J=4.5Hz, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.01 (d, J=5.1 Hz, 2H), 3.23 (m, 2H),2.06 (m, 2H), 1.45 (s, 9H), 1.31 (t, J=7.1 Hz, 3H); ¹³C NMR (CDCl₃, 125MHz) δ 190.9, 170.6, 163.6, 156.1, 151.4, 146.3, 137.6, 134.9, 133.3,130.1, 125.0, 124.9, 123.9, 123.1, 122.3, 121.7, 109.6, 107.2, 79.2,61.6, 45.3, 41.7, 28.4, 14.2; ESIMS m/z (rel intensity) 528 (MNa⁺, 100);HRESIMS calcd for C₂₈H₃₁N₃O₆Na 528.2111 (MNa⁺). found 528.2108 (MNa⁺).

Ethyl2-[(6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino]acetateHydrochloride (40)

Ester 39 (0.060 g, 0.011 mmol) was treated with 2 M HCl in diethyl ether(2 mL) at room temperature for 2 h. The solvent was removed on a rotaryevaporator to yield the solid hydrochloride salt, which was washed with5% MeOH in chloroform to remove impurities and afford the pure solidproduct 40 in quantitative yield: mp 253-254° C. IR (KBr) 3356, 2972,1711, 1689, 1634, 1598, 1544, 1354, 1129, 665 cm⁻¹, ¹H NMR (DMSO-d₆, 300MHz) δ 8.31 (d, J=8.7 Hz, 1H), 8.04 (br s, 3H), 7.64 (m, 1H), 7.47 (m,2H), 7.40 (m, 1H), 7.21 (dd, J=2.2, J=8.7 Hz, 1H), 7.14 (d, J=2.2 Hz,1H), 4.49 (m, 2H), 4.13 (q, J=7.1 Hz, 2H), 4.00 (s, 2H), 2.92 (m, 2H),2.09 (m, 2H), 1.20 (t, J=5.9 Hz, 3H); ¹³C NMR (DMSO-d₆, 300 MHz) δ190.7, 190.2, 170.8, 162.8, 160.6, 156.0, 155.5, 137.3, 136.7, 134.3,130.7, 124.2, 123.1, 118.8, 87.0, 60.4, 56.3, 36.8, 27.6, 18.8, 14.3;ESIMS (m/z, relative intensity) 406 (MH⁺, 100); HRESIMS calcd forC₂₃H₂₃N₃O₄ 406.1767 (MH⁺). found 406.1772 (MH⁺)7: 95.66% (1% TFA inMeOH).

2-[(6-Methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)-amino]aceticAcid (41)

Ethyl2-((6-methyl-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino)acetate(38, 213 mg, 0.59 mmol) was dissolved in methanol (5 mL) andtetrahydrofuran (5 mL). A solution of potassium hydroxide (112 mg, 2.00mmol) in water (5 mL) was added to the reaction mixture, which wasstirred at room temperature for 10 h. Concentrated hydrochloric acid (1mL) was added to the reaction mixture and the solvent removed undervacuum. Water (20 mL) was added, and the mixture was sonicated andfiltered. The solid was washed with water (20 mL). The residue was thendissolved in a mixture of dimethylformamide (0.5 mL) and methanol (9.5mL), heated and allowed to reach room temperature. Ethyl ether (25 mL)and hexane (10 mL) were added and the reaction mixture placed inside therefrigerator overnight. The solvent was filtered off and the residuedried. The product was obtained as a black solid (59 mg, 30.0%): mp 205°C. (dec). IR (KBr) 3361, 3032, 1725, 1698, 1650, 1619, 1578, 1527, 1432,1391, 1318, 1217, 1197, 1054, 1016, 901, 839, 758 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 12.63 (br s, 1H), 8.21 (d, J=8.7 Hz, 1H), 7.68 (d, J=7.5 Hz,1H), 7.43-7.38 (m, 2H), 7.30 (t, J=7.4 Hz, 1H), 7.14 (dd, J=8.8 Hz,J=2.5 Hz, 1H), 7.09 (d, J=2.5 Hz, 1H), 6.56 (br s, 1H), 3.87 (s, 3H),3.86 (s, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 190.7, 172.5, 162.4, 152.7,147.9, 138.3, 134.6, 133.8, 130.5, 124.8, 123.8, 123.4, 122.6, 122.1,121.8, 107.9, 106.5, 44.7, 32.3; negative ion ESIMS m/z (rel intensity)333 [M-H⁺]⁻, 100); HRESIMS calcd for C₁₉H₁₄N₂O₄Na, 357.0851 (MNa⁺).found 357.0857 (MNa⁺); Anal. calcd for C₁₉H₁₄N₂O₄: C, 68.26; H, 4.22; N,8.38. Found: C, 68.03; H, 4.16; N, 8.35.

2-((6-(3-((tert-Butoxycarbonyl)amino)propyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino)aceticAcid (42)

The ester 39 (0.1 g, 0.02 mmol) was dissolved in MeOH-THF (10+10 mL) andaq NaOH (4 N, 5 mL) was added at room temperature. The reaction mixturewas stirred at room temperature for 4 h. The solvent was removed on arotary evaporator and the residue washed with 1 N HCl (30 mL) andextracted with CHCl₃ (2×50 mL). The extract was dried over Na₂SO₄ andconcentrated to afford pure 42 (0.075 g, 80%) which was obtained as abrown solid: mp 190-192° C. IR (KBr) 3350, 2973, 1692, 1645, 1619, 1578,1520, 1364, 1164, 666 cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz) δ 8.30 (d, J=8.7Hz, 1H), 7.59 (m, 1H), 7.45 (m, 2H), 7.39 (m, 1H), 7.21 (dd, J=2.4,J=8.7 Hz, 1H), 7.12 (d, J=2.4 Hz, 1H), 7.07 (m, 1H), 4.40 (m, 2H), 3.90(s, 2H), 3.09 (m, 2H), 1.86 (m, 2H), 1.33 (s, 9H); ESIMS (m/z, relativeintensity) 500 (MNa⁺, 100); HRESIMS calcd for C₂₆H₂₇N₃O₆Na 500.1798(MNa⁺). found 500.1806 (MNa⁺); HPLC purity: 95.5% (MeOH—H₂O, 75:25).

2-((6-(3-Aminopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-3-yl)amino)aceticAcid Hydrochloride (43)

Acid 42 (0.070 g, 0.014 mmol) was treated with HCl in diethyl ether (2M, 2 mL) at room temperature for 1 h. The solvent was removed on arotary evaporator to provide the solid hydrochloride salt, which waswashed with 10% MeOH in chloroform to remove impurities and afford thepure product 43 in quantitative yield: mp 263-264° C. IR (KBr) 3376,2982, 1723, 1695, 1634, 1598, 1566, 1354, 1188, 667 cm⁻¹; ¹H NMR(DMSO-d₆, 300 MHz) δ 8.34 (d, J=7.8 Hz, 1H), 8.28 (s, 1H), 8.12 (br s,3H), 7.64 (m, 1H), 7.49 (m, 2H), 7.39 (m, 1H), 7.19 (dd, J=2.2, J=8.7Hz, 1H), 7.12 (d, J=2.2 Hz, 1H), 4.50 (t, J=6.3 Hz, 2H), 3.90 (s, 2H),2.90 (m, 2H), 2.10 (m, 2H); ¹³C NMR (DMSO-d₆, 125 MHz) δ 190.2, 163.1,162.3, 136.7, 134.1, 130.7, 125.1, 124.2, 124.1, 123.2, 122.6, 107.8,79.2, 41.2, 36.5, 27.2; ESIMS (m/z, relative intensity) 378 (MH⁺, 100);HRESIMS calcd for C₂₁H₁₉N₃O₄ 378.1454 (MH⁺). found 378.1458 (MH⁺); HPLCpurity: 95.3% (1% TFA in MeOH—H₂O, 50:50).

cis-2-(3-Bromopropyl)-3-(3-methoxyphenyl)-7-nitro-1-oxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylicacid (48)

The Schiff base 46 (0,618 g, 2.41 mmol) was diluted in chloroform (50mL) at 0° C. and 4-nitrohomophthalic anhydride (0.5 g, 2.41 mmol) wasadded. The red mixture was stirred at 0° C. for 1 h and then at roomtemperature for 3 h. The creamy orange mixture was filtered and theresidue was washed with CHCl₃ to provide the product as a pale yellowsolid (0.91 g, 82%): mp 122-123° C. IR (film) 3419, 3010, 1714, 1651,1530, 1489, 1346, 1266, 1163, 758 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.60(d, J=2.4 Hz, 1H), 8.25 (dd, J=2.4, J=8.3 Hz, 1H), 7.47 (d, J=8.3 Hz,1H), 7.16 (m, 1H), 6.78 (m, 1H), 6.64 (s, 1H), 6.59 (d, J=7.7 Hz, 1H),5.10 (d, J=2.4 Hz, 1H), 4.08 (m, 1H), 3.65 (s, 3H), 3.33 (m, 2H), 2.92(m, 2H), 2.10 (m, 2H).

6-(3-Bromopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(49)

Thionyl chloride (10 mL) was slowly added to a solution ofcis-4-carboxy-N-(3-chloropropyl)-3,4-dihydro-3-(4-methoxyphenyl)-7-nitro-1(2H)isoquinolone(48, 1.94 g, 4.18 mmol) in benzene (60 mL). The reaction mixture washeated at reflux for 30 min, allowed to cool to room temperature, andconcentrated. The residue was diluted with nitrobenzene (80 mL), chilledin an ice bath, and aluminum chloride (3.00 g, 22.5 mmol) was added. Thereaction mixture was removed from the bath and heated at 100° C. for 1h. Water (200 mL) was added, and the solution was extracted with CHCl₃(3×70 mL). The combined organic layers were washed with sodiumbicarbonate (3×75 mL) and brine (75 mL), and dried over sodium sulfate.The solution was concentrated, hexanes (250 mL) were added, and theliquid was decanted. The solid was washed with hexanes (100 mL) and theliquid was decanted again. The solid was purified by silica gel columnchromatography, eluting with chloroform-methanol, 20:1, to provide ared-orange solid (1.02 g, 55%): mp 282-284° C. (dec). IR (Film) 1699,1667, 1612, 1555, 1499, 1332, 1159, 1078, 840, 785, 688, 662 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 8.85 (d, J=2.5 Hz, 1H), 8.70 (d, J=8.9 Hz, 1H),8.55 (d, J=8.9 Hz, 1H), 7.60 (d, J=8.5 Hz, 1H), 7.38 (s, 1H), 7.06 (d,J=8.3 Hz, 1H), 4.60 (t, J=7.3 Hz, 2H), 3.91 (s, 3H), 3.75 (t, J=6.4 Hz,2H), 2.36 (t, J=7.0 Hz, 2H); EIMS m/z (rel intensity) 444 (M⁺, 100), 442(M⁺, 100), 363 [(M-Br)⁺, 69]; HRESIMS calcd for C₂₀H₁₅BrN₂O₅ 442.0164(MH⁺). found 442.0158 (MH⁺); Anal. Calcd for C₂₀H₁₅BrN₂O₅.0.8H₂O: C,52.49; H, 3.66; N, 6.12; Br, 17.46. Found: C, 52.20; H, 3.54; N, 5.96;Br, 17.46.

6-(3-Azidopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(50)

Sodium azide (0.59 g, 9.07 mmol) and6-(3-bromopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(49; 261 mg, 0.59 mmol) were diluted with DMSO (30 mL), and the mixturewas heated at 90° C. for 12 h. The reaction mixture was diluted withchloroform (100 mL), washed with water (100 mL) and sat aq NaCl (30 mL),and dried over sodium sulfate. The solution was concentrated to providea crude solid that was purified by silica gel column chromatography,eluting with chloroform-methanol, 50:1, to afford an orange solid (92mg, 40%): mp 310° C. (dec). IR (KBr) 3060, 3015, 2979, 2090, 1691, 1668,1614, 1560, 1504, 1371, 1344, 1258, 1232, 1082, 1022, 911, 876, 843, 775cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.88 (d, J=2.5 Hz, 1H), 8.67 (d, J=8.9Hz, 1H), 8.50 (dd, J=8.9 Hz, J=2.5 Hz, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.38(d, J=2.0 Hz, 1H), 7.04 (dd, J=8.1 Hz, J=1.9 Hz, 1H), 4.57 (t, J=7.2 Hz,2H), 3.91 (s, 3H), 3.59 (t, J=6.4 Hz, 2H), 2.07 (t, J=7.0 Hz, 2H); ¹³CNMR (125 MHz, DMSO-d₆) δ 188.6, 164.6, 162.3, 157.7, 145.7, 138.5,136.7, 127.9, 127.3, 125.2, 124.5, 124.3, 123.2, 114.7, 113.7, 108.0,56.6, 49.0, 42.8, 28.3; ESIMS m/z (rel intensity) 405 (MH⁺, 41), 322[(M-C₃H₇N₃)⁺, 100]; HRESIMS m/z calcd for C₂₀H₁₅N₅O₅ 405.1073 (MH⁺).found, 405.1069 (MH⁺); HPLC purity: 95.0% (MeOH, 100), 95.2% (MeOH—H₂O,85:15).

6-(3-Aminopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinolineHydrochloride (51)

Triethylphosphite (1.0 mL) was added to a solution of6-(3-azidopropyl)-5,6-dihydro-8-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(50, 0.287 g, 0.710 mmol) in benzene (50 mL), and the reaction mixturewas heated at reflux for 16 h. The reaction mixture was allowed to coolto room temperature, HCl in methanol (3 M, 10 mL) was added, and thereaction mixture was heated at reflux for 4 h. The reaction mixture wasallowed to cool to room temperature and filtered to provide a red solid(0.180 g, 61%): mp 288-290° C. IR (KBr) 3060, 3015, 2979, 2090, 1691,1668, 1614, 1560, 1504, 1371, 1344, 1258, 1232, 1082, 1022, 911, 876,843, 775 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.84 (d, J=2.5 Hz, 1H), 8.69(d, J=8.9 Hz, 1H), 8.53 (dd, J=8.9 Hz, J=2.5 Hz, 1H), 7.87 (br s, 3H),7.60 (d, J=8.4 Hz, 1H), 7.30 (d, J=2.0 Hz, 1H), 7.05 (dd, J=8.1 Hz,J=1.9 Hz, 1H), 4.54 (t, J=7.2 Hz, 2H), 3.91 (s, 3H), 2.94 (t, J=6.4 Hz,2H), 2.10 (t, J=7.0 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.6, 164.6,162.6, 157.6, 145.8, 138.3, 136.7, 128.0, 127.3, 125.3, 124.6, 124.2,123.2, 114.6, 113.8, 108.2, 56.6, 42.3, 37.2, 27.3; ESIMS m/z (relintensity) 380 (MH⁺, 72), 363 ([(M-NH₃]⁺, 100); HRESIMS m/z calcd forC₂₀H₁₇N₃O₅ 380.1246 (MH⁺). found, 380.1243; HPLC purity: 97.2%(MeOH—H₂O, 90:10).

6-(3-(1H-Imidazol-1-yl)propyl)-8-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(52)

6-(3-Bromopropyl)-8-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(49, 120 mg, 0.27 mmol) was dissolved dimethylformamide (2 mL) anddioxane (10 mL). Sodium iodide (208 mg, 1.40 mmol) was added and thereaction mixture was heated at 60° C. for 6 h. Imidazole (202 mg, 297mmol) and potassium carbonate (225 mg, 1.63 mmol) were added and thereaction mixture was stirred at 90° C. for 12 h. Water (15 mL) was addedand a precipitated formed. The solid was filtered and kept. Theremaining solution was diluted with water (100 mL) and the aqueous phaseextracted with chloroform (3×30 mL). The organic extracts were combined,washed with water (3×100 mL), and dried over sodium sulfate. Thefiltered solid from the previous step was combined with the organicextracts, the solvent was removed in vacuo and the compound purified bysilica gel column chromatography, eluting with chloroform-methanol,50:1. The compound was obtained as a dark yellow solid (47 mg, 40%): mp265-276° C. IR (Film) 1693, 1673, 1614, 1559, 1336, 1234, 1075, 866,842, 774 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 9.19 (d, J=2.1 Hz, 1H), 8.89(d, J=8.9 Hz, 1H), 8.48 (dd, J=8.9 Hz, J=2.2 Hz, 1H), 7.68-7.60 (m, 2H),7.13 (br s, 1H), 7.06 (br s, 1H), 6.86-6.83 (m, 2H), 4.55 (t, J=7.1 Hz,2H), 4.23 (t, J=6.5 Hz, 2H), 3.93 (s, 3H), 2.41 (p, J=7.1 Hz, 2H); ¹³CNMR (CDCl₃, 125 MHz) δ 188.6, 164.3, 162.4, 157.6, 145.4, 137.9, 136.5,136.1, 128.2, 126.8, 125.4, 124.4, 124.2, 122.9, 114.2, 113.7, 107.9,56.5, 46.5, 41.7, 29.7; ESIMS m/z (rel intensity) 431 (MH⁺, 100);HRESIMS calcd for C₂₃H₁₈N₄O₅ 431.1355 (MH⁺). found 431.1352 (MH⁺); HPLCpurity: 96.8% (MeOH—H₂O, 90:10).

8-Methoxy-6-(3-morpholinopropyl)-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(53)

6-(3-Bromopropyl)-8-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(49, 117 mg, 0.26 mmol) was dissolved in dimethylformamide (2 mL) anddioxane (10 mL). Sodium iodide (213 mg, 1.42 mmol) was added and thereaction mixture was heated at 60° C. for 6 h. Morpholine (0.2 mL, 2.30mmol) and potassium carbonate (260 mg, 1.88 mmol) were added and thereaction mixture was stirred at 90° C. for 12 h. Water (200 mL) wasadded and the compound extracted with chloroform (3×35 mL). The organicextracts were combined, washed with water (3×100 mL) and dried oversodium sulfate. The solvent was removed in vacuo and the compoundpurified by silica gel column chromatography, eluting withchloroform-methanol, 50:1. The product was obtained as an orange solid(30 mg, 25%): mp 276-278° C. IR (Film) 3020, 1676, 1614, 1562, 1337,1215, 1066, 846, 757, 666 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 9.18 (d, J=2.2Hz, 1H), 8.90 (d, J=8.9 Hz, 1H), 8.47 (dd, J=9.0 Hz, J=2.3 Hz, 1H),7.50-7.48 (m, 2H), 7.10 (dd, J=8.9 Hz, J=2.1 Hz, 1H), 4.63 (t, J=7.6 Hz,2H), 4.03 (s, 3H), 3.72 (m, 4H), 2.59 (t, J=6.2 Hz, 2H), 2.50 (s, 4H)2.06 (m, 2H); ¹³C NMR (CDCl₃, 125 MHz) δ 188.0, 162.5, 157.5, 156.9,145.3, 137.8, 136.8, 136.7, 128.2, 124.2, 123.1, 118.6, 118.5, 117.9,106.9, 63.5, 56.4, 53.5, 51.3, 42.0, 23.5; ESIMS m/z (rel intensity) 450(MH⁺, 62); HRESIMS calcd for C₂₄H₁₃N₃O₆ 450.1665 (MH⁺). found 450.1660(MH⁺); HPLC purity: 97.7% (MeOH—H₂O, 85:15), 97.8% (MeOH—H₂O, 90:10).

1-Bromo-6-cyanophthalide (65)

6-Cyanophthalide (64, 2.73 g, 17.2 mmol) was dissolved in carbontetrachloride (120 mL). 3-Chloroperbenzoic acid (100 mg) andN-bromosuccinimide (3.20 g, 17.9 mmol) were added. Light was appliedwith a 250 W lamp and the reaction mixture was heated at reflux for 24h. The solvent was removed and the residue purified by silica gel columnchromatography, eluting with ethyl acetate-hexane, 1:7. The product wasobtained as a white solid (2.32 g, 56.7%): mp 132-134° C. IR (Film)3090, 3064, 3032, 2992, 2235, 1788, 1738, 1428, 1295, 1227, 1119, 990,717, 664 cm⁻¹; NMR (300 MHz, CDCl₃) δ 8.23 (s, 1H), 8.05 (dd, J=1.1 Hz,J=8.1 Hz, 1H), 7.79 (d, J=7.9 Hz, 1H), 7.44 (s, 1H); ¹³C NMR (75 MHz,CDCl₃) δ 165.1, 152.2, 138.3, 130.0, 125.0, 121.5, 116.8, 115.3, 73.4;CIMS m/z (rel intensity) 238 (MH⁺, 13), 160 [(MH—Br)⁺, 100].

7-Cyano-3-hydroxyphthalide (66)

1-Bromo-6-cyanophthalide (65, 1.99 g, 8.36 mmol) was dissolved in hotwater (50 mL). The reaction mixture was heated at reflux for 3 h.Ethanol (20 mL) was added and the solution was concentrated to half itsvolume and heated at reflux for 5 min. Once the solution reached roomtemperature the reaction mixture was placed in the refrigerator. Thedesired compound was recrystallized as off-white solid (1.23 g, 84.1%):mp 138-140° C. ¹H NMR (300 MHz, DMSO-d₆) δ 8.42 (br s, 1H), 8.37 (d,J=1.8 Hz, 1H), 8.24 (dd, J=7.8 Hz, J=1.7 Hz, 1H), 7.90 (d, J=7.9 Hz,1H), 6.75 (s, 1H); ESIMS m/z (rel intensity) 174 (MH⁺, 100).

3-Cyano-5,11-dihydro-5,11-dioxoindeno[1,2-c]isochromene (68)

7-Cyano-3-hydroxyphthalide (66, 1.41 g, 8.06 mmol) and phthalide (67,1.08 g, 8.06 mmol) were dissolved in ethyl acetate (15 mL). Sodium (0.80g, 34.8 mmol) was added to methanol (30 mL) at 0° C. and the resultingsolution added to the reaction mixture. The reaction mixture was stirredfor 30 min at room temperature and then heated at reflux for 6 h.Concentrated hydrochloric acid (15 mL) was added and the solvent removedunder vacuum. Benzene (100 mL) and para-toluenesulfonic acid (50 mg)were added, and the reaction mixture was connected to a Dean-Stark trapand heated at reflux for 16 h. Chloroform (150 mL) was added and thereaction mixture was heated at reflux and immediately filtered. Thesolvent was removed under vacuum and the solid recrystallized fromchloroform to remove unreacted phthalide. The compound was purified bysilica gel column chromatography, eluting with chloroform-methanol,50:1. The product was obtained as a yellow solid (1.03 g, 46.8%): mp225-227° C. IR (KBr) 3118, 3082, 2232, 1760, 1747, 1715, 1503, 1385,1309, 1018, 877, 777, 763, 665 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.59(s, 1H), 8.47 (d, J=8.2 Hz, 1H), 7.99 (dd, J=8.2 Hz, J=1.6 Hz, 1H), 7.64(d, J=7.9 Hz, 1H), 7.55-7.50 (m, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ188.8, 172.6, 158.8, 137.9, 135.8, 135.4, 135.1, 134.0, 132.7, 124.1,123.6, 120.6, 119.2, 117.3, 11.7, 106.5; CIMS m/z (rel intensity) 274(MH⁺, 100).

3-Cyano-5,11-dihydro-6-[3-(1H-imidazolyl)propyl]-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]isoquinoline(72)

3-Cyano-5,11-dihydro-5,11-dioxoindeno-[1,2-c]isochromene (68, 200 mg,0.73 mmol) was dissolved in tetrahydrofuran (10 mL).3-(1H-Imidazol-1-yl)propyl-1-amine (69, 335 mg, 2.67 mmol) was dissolvedin chloroform (5 mL) and the solution added to the reaction mixture. Thereaction mixture was stirred for 3 h at room temperature and then heatedat reflux for 1 h. The solution turned red and, upon reflux, an orangeprecipitate formed. The solvent was removed under vacuum and the solidpurified by silica gel column chromatography, eluting withchloroform-methanol, 30:1. The desired compound was obtained as a yellowsolid (103 mg, 37%): mp 256-258° C. IR (KBr) 3103, 2952, 2225, 1694,1671, 1611, 1534, 1509, 1433, 1192, 850, 764, 665 cm⁻¹; ¹H NMR (300 MHz,DMSO-d₆) δ 8.80 (d, J=8.6 Hz, 1H), 8.63 (d, J=1.4 Hz, 1H), 7.88 (dd,J=8.4 Hz, J=1.6 Hz, 1H), 7.67-7.64 (m, 2H), 7.43 (t, J=7.6 Hz, 1H), 7.35(dt, J=7.5 Hz, J=1.2 Hz, 1H), 7.21 (s, 1H), 7.07 (s, 1H), 6.66 (d, J=7.5Hz, 1H), 4.54 (t, J=7.8 Hz, 2H), 4.26 (t, J=6.2 Hz, 2H), 2.37 (p, J=8.2Hz, J=6.6 Hz, 2H); ESIMS m/z (rel intensity) 381 (MH⁺, 100); HRESIMScalcd for C₂₃H₁₆N₄O₂ 381.1352 (MH⁺). found 381.1345 (MH⁺); HPLC purity:95.0% (MeOH—H₂O, 85:15), 95.4% (MeOH—H₂O, 95:5).

3-Cyano-6,11-dihydro-5,11-dioxo-6-(3-morpholinylpropyl)-5H-indeno-[1,2-c]isoquinoline(73)

3-Cyano-5,11-dihydro-5,11-dioxoindeno[1,2-c]isochromene (68, 130 mg,0.47 mmol) was dissolved in chloroform (50 mL).3-Morpholinopropyl-1-amine (70, 152 mg, 1.04 mmol) and molecular sieveswere added and the reaction was mixture stirred at room temperature for8 h. The reaction mixture was then heated at reflux for 1 h. Themolecular sieves were filtered off, the solvent removed under vacuum andthe residue purified by silica gel column chromatography, eluting withchloroform-methanol, 100:1. The desired compound was obtained as anorange solid (113 mg, 60.0%): mp 259-261° C. IR (Film) 3083, 2950, 2849,2223, 1698, 1673, 1611, 1509, 1435, 1275, 1185, 1116, 998, 959, 896,851, 766, 665 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.80 (d, J=8.6 Hz, 1H),8.62 (d, J=1.7 Hz, 1H), 7.88-7.84 (m, 2H), 7.69 (m, 1H), 7.51-7.48 (m,2H), 4.64 (t, J=7.8 Hz, 2H), 3.70 (t, J=4.4 Hz, 4H), 2.58 (t, J=6.2 Hz,1H), 2.49 (br s, 4H), 2.05 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 189.5,162.0, 157.8, 136.3, 135.3, 135.0, 133.4, 131.9, 124.4, 123.6, 123.4,123.0, 110.0, 107.2, 66.9, 56.0, 53.9, 43.9, 25.7; ESIMS m/z (relintensity) 400 (MH⁺, 100); HRESIMS calcd for C₂₄H₂₁N₃O₃ 400.1661 (MH⁺).found, 400.1665 (MH⁺). Anal. calcd for C₂₄H₂₁N₃O₃.0.5H₂O: C, 70.57; H,5.43; N, 10.29. Found: C, 70.71; H, 5.22; N, 10.01.

3-Cyano-6,11-dihydro-5,11-dioxo-6-[3-(dimethylaminopropyl]-5H-indeno[1,2-c]isoquinoline(74)

3-Cyano-5,11-dihydro-5,11-dioxoindeno[1,2-c]isochromene (68, 135 mg,0.49 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL).N′,N′-Dimethylpropane-1,3-diamine (71, 158 mg, 1.51 mmol) was dissolvedin chloroform (5 mL). This solution was added to the first solution, andthe reaction mixture was stirred for 3 h at room temperature. Themixture was heated at reflux for 1 h. The solvent was removed undervacuum and the solid purified by silica gel column chromatography,eluting with chloroform-methanol, 50:1. The compound was obtained as ayellow solid (96 mg, 55%): mp 210-212° C. IR (Film) 2817, 2762, 2223,1698, 1673, 1611, 1611, 1435, 1261, 1191, 1040, 897, 764 cm⁻¹; ¹H NMR(500 MHz, DMSO-d₆) δ 8.78 (d, J=8.5 Hz, 1H), 8.62 (s, 1H), 7.87-7.84 (m,2H), 7.67 (d, J=6.7 Hz, 1H), 7.50-7.46 (m, 2H), 4.59 (t, J=5.9 Hz, 2H),2.53 (t, J=6.5 Hz, 2H), 2.32 (s, 6H), 2.04 (br p, J=8.5 Hz, 2H); ¹³C NMR(125 MHz, DMSO-d₆) δ 189.6, 162.0, 157.9, 136.3, 135.3, 135.1, 135.0,133.6, 133.5, 131.8, 124.4, 123.7, 123.5, 123.0, 118.2, 109.9, 107.3,56.6, 45.6, 43.8, 29.9; ESIMS m/z (rel intensity) 402 (MH⁺, 100);HRESIMS calcd for C₂₃H₁₉N₃O₄ 402.1454 (MH⁺). found 402.1452 (MH⁺); HPLCpurity: 97.4% (MeOH—H₂O, 95:5), 97.4% (MeOH).

3-Cyano-5,11-dihydro-8,9-methylenedioxy-5,11-dioxo[1,2-c]isochromene(76)

7-cyano-3-hydroxyphthalide (66, 1.55 g, 8.85 mmol) and5,6-methylenedioxyphthalide (75, 1.58 g, 8.06 mmol) were dissolved inethyl acetate (20 mL). Sodium (0.85 g, 34.8 mmol) was dissolved inmethanol (35 mL) at 0° C. and the resulting solution added to thereaction mixture. The reaction mixture was stirred for 30 min at roomtemperature and then heated at reflux for 15 h. Concentratedhydrochloric acid (20 mL) was added and the solvent removed undervacuum. Benzene (100 mL) and para-toluenesulfonic acid (50 mg) wereadded, and the reaction mixture was connected to a Dean-Stark trap andheated at reflux for 16 h. Chloroform (100 mL) was added, the reactionmixture heated at reflux and immediately filtered. The solvent wasremoved under vacuum and the solid recrystallized fromchloroform-methanol, 25:1, to remove unreacted phthalide. The compoundwas purified by silica gel column chromatography, eluting withchloroform-methanol, 50:1. The product was obtained as a yellow solid(133 mg, 10%): mp>350° C. Also, 0.81 g of the starting material 75 wasrecovered. IR (Film) 2233, 1692, 1417, 1313, 1152, 1029, 925, 861, 785,665 cm⁻¹; ¹H NMR (CDCl₃-MeOH-d₄, 300 MHz) δ 8.44 (s, 1H), 8.29 (d, J=8.1Hz, 1H), 7.88 (dd, J=8.1 Hz, J=1.5 Hz, 1H), 7.05 (s, 1H), 6.94 (s, 1H),6.09 (s, 2H); EIMS m/z (rel intensity) 317 (M⁺, 100); HREIMS calcd forC₁₈H₇NO₅ 317.0324 (M⁺). found 317.0327 (M⁺).

3-Cyano-5,11-dihydro-6-[3-(dimethylamino)propyl]-5,11-dioxoindeno-[1,2-c]isoquinoline(77)

3-Cyano-5,11-dihydro-8,9-methylenedioxy-5,11-dioxo[1,2-c]isochromene(76, 91 mg, 0.28 mmol) was dissolved in tetrahydrofuran (25 mL).N¹,N¹-Dimethylpropane-1,3-diamine (71, 156 mg, 1.56 mmol) and molecularsieves were added and the reaction mixture stirred at room temperaturefor 8 h. The reaction mixture was then heated at reflux for 1 h. Themolecular sieves were filtered off, the solvent removed in vacuo and theresidue purified by recrystallization from chloroform-hexane. Thedesired compound was obtained as a purple solid (89 mg, 79%): mp280-282° C. IR (Film) 2968, 2909, 2825, 2271, 1694, 1672, 1607, 1577,1529, 1432, 1309, 1282, 1185, 1033, 852, 793 cm⁻¹; ¹H NMR (500 MHz,CDCl₃-MeOH-d₄) δ 8.62 (d, J=8.5 Hz, 1H), 8.51 (s, 1H), 7.86 (d, J=8.5Hz, 1H), 7.44 (s 1H), 7.08 (s, 1H), 6.08 (s, 1H), 4.45 (t, J=6.9 Hz,2H), 2.46 (br s, 2H), 2.25 (s, 6H), 1.94 (m, 2H); ¹³C NMR (125 MHz,CDCl₃) δ 188.7, 162.2, 157.6, 152.0, 150.0, 135.2, 133.6, 131.3, 130.7,123.8, 122.1, 118.1, 109.1, 106.6, 106.3, 105.4, 102.9, 56.4, 45.4,43.5, 33.0, 26.8; ESIMS m/z (rel intensity) 401 (M⁺, 100); HPLC purity:100% (MeOH—H₂O, 90:10), 96.7% (MeOH—H₂O, 85:15).

6-(3-Bromopropyl)-9-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(80)

5-Nitrohomophthalic anhydride (47, 5.02 g, 24.2 mmol) was added tosolution of 3-bromo-N-(4-methoxybenzylidene)propyl-1-amine (78, 6.21 g,24.2 mmol) in chloroform (115 mL), and the reaction mixture was allowedto stir at room temperature for 1.5 h and then placed inside the freezerovernight. The precipitate was filtered and washed with chloroform (50mL) and dried to provide intermediate 79 (5.13 g), which was usedwithout further purification. Thionyl chloride (26 mL) was added to asuspension of 79 in benzene (200 mL) and the reaction mixture was heatedat reflux until complete dissolution. The solvent was removed undervacuum and the residue diluted with nitrobenzene (225 mL) and chilled inan ice bath. Aluminum chloride (4.50 g, 33.8 mmol) was added and thereaction mixture was heated at 100° C. for 2 h. Cool water (200 mL) wasadded, the organic phase separated and the aqueous phase was extractedwith CHCl₃ (3×70 mL). The combined organic layer was washed with asolution of NaHCO₃ (4.0 g) in water (75 mL). The solution wasconcentrated and hexanes were added until the precipitation of a redsolid was observed. The solid was filtered, washed with hexanes (100mL), and purified by flash silica gel column chromatography, elutingwith chloroform-methanol, 12:1. The product was obtained as an orangesolid (0.79 g, 11.2%): mp 282-284° C. IR (Film) 3097, 1809, 1745, 1685,1604, 1510, 1337, 1231, 1127, 1082, 851 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ9.17 (d, J=2.3 Hz, 1H), 8.81 (d, J=8.9 Hz, 1H), 8.47 (dd, J=9.0 Hz,J=2.3 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 7.28 (d, J=2.5 Hz, 1H), 6.94 (dd,J=8.5 Hz, J=2.5 Hz, 1H), 4.51 (t, J=6.0 Hz, 2H), 3.84 (s, 3H), 3.75 (dd,J=7.5 Hz, J=6.6 Hz, 2H), 2.30 (br p, J=8.5 Hz, 2H); EIMS m/z (relintensity) 442 (M⁺, 100); HREIMS calcd for C₂₀H₁₅N₂O₅Br 442.0164 (M⁺).found, 442.0158 (M⁺).

3-Amino-6-(3-bromopropyl)-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(81)

6-(3-Bromopropyl)-9-methoxy-3-nitro-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(80, 1.00 g, 2.26 mmol) was dissolved in tetrahydrofuran (60 mL),methanol (20 mL), and ethyl acetate (30 mL) and transferred to a Parrshaker flask. The reaction vessel was purged with argon for 10 min andthen palladium-charcoal (10%, 20 mg) was added. The reaction mixture wasshaken for 24 h under an atmosphere of hydrogen (60 psi). The solid wasfiltered off, the solvent removed under vacuum, and the compoundpurified by silica gel column chromatography, eluting with ethylacetate. The product was obtained as a brown solid (0.55 g, 59%): mp330° C. (dec). IR (Film) 3367, 1696, 1655, 1506, 1480, 1431, 1293, 1229,1016, 834, 787, 665 cm⁻¹; ¹H NMR (300 MHz, DMSO-d₆) δ 8.35 (d, J=8.6 Hz,1H), 7.67-7.62 (m, 2H), 7.06 (d, J=2.5 Hz, 1H), 7.0.1-6.94 (m, 2H), 4.51(t, J=6.0 Hz, 2H), 3.84 (s, 3H), 3.75 (dd, J=7.5 Hz, J=6.6 Hz, 2H), 2.30(br p, J=8.5 Hz, 2H); EIMS m/z (rel intensity) 413 (M⁺, 46), 333([M-HBr)]⁺, 100); HRESIMS m/z calcd for C₂₀H₁₇N₂O₃Br 413.0501 (MH⁺).found, 413.0505 (MH⁺).

6-(3-Bromopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(82)

3-Amino-6-(3-bromopropyl)-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(81, 420 mg, 1.01 mmol) was dissolved in dioxane (10 mL). Concentratedhydrochloric acid (0.65 mL, 3.3 mmol) was added and the reaction mixturewas stirred for 10 min and then cooled down to 0° C. A solution ofsodium nitrite (114 mg, 1.65 mmol) in water (5 mL) was slowly added withstirring while the temperature was −20° C. The reaction mixture wasstirred for 30 min and then slowly added to a solution of copper (I)iodide (200 mg) and potassium iodide (300 mg) in water (15 mL). Thereaction mixture was stirred at room temperature for 8 h and then heatedat reflux for 1 h. The reaction mixture was diluted with water (200 mL)and extracted with chloroform (3×100 mL). The combined organic extractswere washed with an aqueous solution of sodium thiosulfate (2×200 mL),aqueous sodium bicarbonate (2×200 mL), water (100 mL) and brine (100mL). The organic solution was dried over sodium sulfate and the solventremoved under vacuum. The residue was purified by silica gel columnchromatography, eluting with chloroform-methanol, 30:1. Compound 82 wasobtained as a dark red solid (402 mg, 76%): mp 207-209° C. IR (Film)2922, 1765, 1655, 1599, 1532, 1478, 1455, 1430, 1379, 1295, 1225, 1047,969, 812, 788, 691, 666, 592 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.60 (d,J=1.5 Hz, 1H), 8.32 (d, J=8.6 Hz, 1H), 7.93 (dd, J=8.5 Hz, J=1.5 Hz,1H), 7.64 (d, J=8.4 Hz, 1H), 7.15 (d, J=2.3 Hz, 1H), 6.82 (dd, J=8.3 Hz,J=2.4 Hz, 1H), 4.58 (t, J=7.2 Hz, 2H), 3.89 (s, 3H), 3.64 (t, J=6.2 Hz,2H), 2.44 (t, J=7.2 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 189.3, 162.5,162.0, 156.6, 142.4, 137.6, 137.0, 131.4, 127.6, 124.7, 124.1, 123.9,115.8, 111.1, 107.6, 91.2, 55.8, 44.2, 31.1, 30.2; ESIMS m/z (relintensity) 523 (MH⁺, 60), 403 [(MH⁺—C₃H₆Br)⁺, 100]; HRESIMS m/z calcdfor C₂₀H₁₅NO₃IBr 523.9358 (MH⁺). found, 523.9362 (MH⁺).

6-(3-Azidopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(83)

Sodium azide (0.59 g, 9.07 mmol) and6-(3-bromopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(82, 261 mg, 0.59 mmol) were diluted with DMSO (30 mL) and the mixturewas heated at 90° C. for 12 h. The reaction mixture was diluted withCHCl₃ (100 mL), washed with water (100 mL) and sat aq NaCl (30 mL), anddried over sodium sulfate. The solution was concentrated to provide acrude solid that was purified by silica gel column chromatography,eluting with chloroform-methanol, 50:1, to afford an orange solid (0.16g, 83%): mp 267-269° C. (dec). IR (KBr) 2090, 1688, 1662, 1598, 1532,1478, 1424, 1298, 1164, 1014, 819, 791, 694 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ 8.61 (d, J=1.8 Hz, 1H), 8.34 (d, J=8.6 Hz, 1H), 7.92 (dd, J=8.6Hz, J=1.8 Hz, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.17 (d, J=2.5 Hz, 1H), 6.85(dd, J=8.3 Hz, J=2.8 Hz, 1H), 4.53 (t, J=7.2 Hz, 2H), 3.89 (s, 3H), 3.63(t, J=6.1 Hz, 2H), 2.11 (t, J=7.2 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃) δ188.9, 162.5, 161.8, 156.5, 142.2, 137.7, 136.9, 131.3, 127.6, 124.6,123.9, 123.8, 115.8, 110.9, 107.4, 91.0, 55.7, 49.2, 42.7, 28.3; ESIMSm/z (rel intensity) 509 (MNa⁺, 100); HRESIMS m/z calcd for C₂₀H₁₅N₄O₃I487.0267 (MH⁺). found, 487.0271 (MH⁺).

6-(3-Aminopropyl)-5,6-dihydro-9-methoxy-3-iodo-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(84)

Triethylphosphite (0.2 mL) was added to a solution of6-(3-azidopropyl)-3-iodo-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(83, 0.186 g, 0.459 mmol) in benzene (30 mL), and the reaction mixturewas heated at reflux for 16 h. The reaction mixture was allowed to coolto room temperature, HCl in methanol (prepared from 0.5 mL of acetylchloride in 9.5 mL of methanol) was added, and the reaction mixture washeated at reflux for 4 h. The reaction mixture was allowed to cool toroom temperature and filtered to provide a red solid (0.159 g, 83%): mp267-269° C. (dec). IR (KBr) 3399, 2937, 1692, 1658, 1600, 1532, 1478,1429, 1298, 1228, 909, 823, 733 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ 8.65 (d,J=1.5 Hz, 1H), 8.37 (d, J=8.5 Hz, 1H), 7.94 (dd, J=8.6 Hz, J=1.6 Hz,1H), 7.64 (d, J=8.4 Hz, 1H), 7.21 (d, J=2.4 Hz, 1H), 6.84 (dd, J=8.3 Hz,J=2.4 Hz, 1H), 4.58 (t, J=7.2 Hz, 2H), 3.89 (s, 3H), 2.89 (t, J=6.3 Hz,2H), 2.00 (t, J=7.3 Hz, 2H); ESIMS m/z (rel intensity) 461 (MH⁺, 100);HRESIMS m/z calcd for C₂₀H₁₇N₂O₃I 461.0362 (MH⁺). found, 461.0371 (MH⁺);HPLC purity: 98.9% (MeOH—H₂O, 90:10), 96.7% (MeOH—H₂O, 80:20).

3-Iodo-9-methoxy-6-(3-(Dimethylamino)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(85)

3-Amino-6-(3-aminopropyl)-9-methoxy-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione(84, 111 mg, 1.93 mmol) was dissolved in methanol (10 mL) and aceticacid (5 mL). Aqueous formaldehyde (37%, 0.15 mL) was added and thereaction mixture was stirred at room temperature for 2 h. The reactionmixture was cooled to 0° C. and sodium cyanoborohydride (300 mg) wasslowly added. The reaction mixture was stirred for 1 h at roomtemperature. Water (30 mL) was added and the reaction mixture extractedwith chloroform (2×25 mL). The organic extracts were combined and washedwith brine (30 mL). The solvent was removed in vacuo and the compoundpurified by silica gel column chromatography, eluting with chloroform.The product was obtained as a red solid (65 mg, 55%) mp>350° C. IR(Film) 3054, 2987, 1698, 1649, 1531, 1477, 1430, 1301, 1265, 740 cm⁻¹;¹H NMR (500 MHz, CDCl₃) δ 8.65 (d, J=1.8 Hz, 1H), 8.38 (d, J=8.6 Hz,1H), 7.85 (dd, J=2.0, J=8.6 Hz, 1H), 7.67 (d, J=8.3 Hz, 1H), 7.21 (d,J=2.5 Hz, 1H), 6.83 (dd, J=2.5, J=8.4 Hz, 1H), 4.54 (t, J=8.1 Hz, 2H),3.89 (s, 3H), 2.50 (d, J=6.3 Hz, 2H), 2.02 (m, 2H); ¹³C NMR (125 MHz,CDCl₃-MeOH-d₄) δ 189.5, 162.6, 162.2, 142.4, 137.7, 136.9, 131.6, 127.6,124.6, 124.3, 123.8, 115.9, 111.2, 107.7, 91.0, 56.2, 55.7, 44.7, 43.0,26.4; ESIMS m/z (rel intensity) 489 (MH⁺, 100); HRESIMS m/z calcd forC₂₂H₂₁N₂O₃I 489.0675 (MH⁺). found, 489.0670 (MH⁺); HPLC purity: 97.1%(MeOH—H₂O, 95:5); 97.5% (MeOH—H₂O, 85:15).

3-Amino-6-(3-chloropropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(87)

6-(3-Chloropropyl)-5,6-dihydro-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(86) (0.220 g, 0.552 mmol) and 5% Pd/C (0.200 g) were diluted with THF(125 mL). The solution was degassed and allowed to stir at roomtemperature under a hydrogen atmosphere for 1 h. The solution wasfiltered, the filterpad was washed with chloroform (125 mL), and thefiltrate was concentrated to provide a crude purple solid. The solid waspurified by silica gel flash column chromatography, eluting withchloroform to provide a purple solid (0.080 g, 39%): mp 232-235° C.(dec). IR (KBr) 3357, 1644, 1544, 1510, 1272, 1052 cm⁻¹; ¹H NMR(DMSO-d₆) δ 8.33 (d, J=8.6 Hz, 1H), 7.42 (dd, J=8.5 Hz, J=6.8 Hz, 1H),7.32 (m, 2H), 7.14 (dd, J=6.7 Hz, J=1.0 Hz, 1H), 7.09 (dd, J=8.7 Hz,J=2.4 Hz, 1H), 5.74 (s, 2H), 4.68 (t, J=7.1 Hz, 2H), 3.97 (s, 3H), 3.73(t, J=6.6 Hz, 2H), 2.21 (pent, J=7.1 Hz, 2H); ESIMS m/z (rel intensity)369/371 (MH⁺, 100/32). Anal. calcd for C₂₀H₁₇ClN₂O₃: C, 65.13; H, 4.65;N, 7.60. Found: C, 64.85; H, 4.74; N, 7.42.

3-Amino-6-(3-azidopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(88)

Sodium azide (0.063 g, 0.968 mmol) and3-amino-6-(3-chloropropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(87, 0.119 g, 0.323 mmol) were diluted with DMSO (25 mL) and the mixturewas heated at 100° C. for 16 h. The reaction mixture was diluted withchloroform (50 mL), washed with water (3×25 mL), brine (25 mL), anddried over sodium sulfate. The solution was concentrated to provide acrude solid that was purified by silica gel column chromatography,eluting with a gradient of chloroform to 1% MeOH-chloroform, to providea solid that was washed with diethyl ether to afford a purple solid(0.102 g, 84%): mp 220-223° C. IR (KBr) 3434, 3349, 2096, 1651, 1509,1271 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.34 (d, J=8.7 Hz, 1H), 7.42 (dd, J=8.5Hz, J=6.7 Hz, 1H), 7.33 (m, 2H), 7.15 (dd, J=6.7 Hz, J=1.1 Hz, 1H), 7.09(dd, J=8.7 Hz, J=2.5 Hz, 1H), 5.74 (s, 2H), 4.65 (m, 2H), 3.97 (s, 3H),3.46 (t, J=6.7 Hz, 2H), 1.99 (m, 2H); ESIMS m/z (rel intensity) 376(MH⁺, 55). Anal. calcd for C₂₀H₁₇N₅O₃: C, 63.99; H, 4.56; N, 18.66.Found: C, 63.78; H, 4.38; N, 18.30.

3-Amino-6-(3-aminopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinolineDihydrochloride (89)

Triethyl phosphite (0.07 mL) was added to a solution of3-amino-6-(3-azidopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline(88, 0.061 g, 0.163 mmol) in benzene (20 mL) and the reaction mixturewas heated at reflux for 16 h. The reaction mixture was allowed to coolto room temperature, 3 M HCl in methanol (10 mL) was added, and thereaction mixture was heated at reflux for 3 h. The reaction mixture wasallowed to cool to room temperature, concentrated, and the precipitatewas washed with chloroform (50 mL) and filtered to provide an orangesolid (0.054 g, 78%): mp 246-248° C. (dec). IR (KBr) 3445, 2929, 1651,1544, 1505, 1479, 1266 cm⁻¹; ¹H NMR (DMSO-d₆) δ 8.51 (d, J=8.7 Hz, 1H),8.03 (bs, 2H), 7.77 (d, J=2.2 Hz, 1H), 7.49 (m, 2H), 7.36 (d, J=7.6 Hz,1H), 7.19 (dd, J=6.8 Hz, J=0.8 Hz, 1H), 4.61 (t, J=7.0 Hz, 2H), 4.01 (s,3H), 2.86 (m, 2H), 2.09 (m, 2H); ESIMS m/z (rel intensity) 350 (MH⁺,90). Anal. calcd for C₂₀H₂₁Cl₂N₃O₃.1.0H₂O: C, 54.55; H, 5.26; N, 9.54.Found: C, 54.87; H, 5.10; N, 9.21.

Molecular Modeling.

The Tdp1 crystal structure (PDB: 1RFF) was prepared by removing one ofthe monomers along with all crystallized waters, thepolydeoxyribonucleotide 5′-D(*AP*GP*TP*T)-3′, the Top1-derived peptideresidues 720-727 (mutation L724Y), and all metal ions. The Lys265,Lys495 and His493 residues were protonated. Missing hydrogens were addedas needed. GOLD docking was performed using the centroid x=8.0128,y=46.5888, z=−1.5534. The hydrogen bond length was set to 4 Å while thevan der Waals parameter was set to 10 Å. The top ligand binding-pose(highest GOLD score) was selected and merged with the prepared protein.The ligand was surrounded by a sphere of 6 Å of radius and minimized bythe conjugate gradient method using the MMFF94s force field and MMFF94charges with Sybyl software. The calculation was terminated when thegradient reached a value of 0.05 kcal/(mol·Å).

Topoisomerase I-Mediated DNA Cleavage Reactions.

Top1 reactions were performed as recently described. Briefly, a3′-[³²P]-end-labeled 117-bp DNA oligonucleotide (Integrated DNATechnologies) was incubated at 2 nM with recombinant Top1 in 20 μL ofreaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mMEDTA, and 15 μg/mL BSA] at 25° C. for 20 min in the presence of variousconcentrations of compounds. The reactions were terminated by adding SDS(0.5% final concentration) followed by the addition of two volumes ofloading dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA,0.1% xylene cyanol, and 0.1% bromphenol blue). Reactions were subjectedto 20% denaturing PAGE. Gels were dried and visualized by using aTyphoon 8600 and ImageQuant software (Molecular Dynamics).

Gel-based Assay Measuring the Inhibition of Recombinant Tdp1.

Tdp1 reactions were performed as recently described. Briefly, a5′-[³²P]-labeled single-stranded DNA oligonucleotide containing a3′-phosphotyrosine (N14Y) incubated with 5 pM recombinant Tdp1 in theabsence or presence of inhibitor for 15 min at room temperature in abuffer containing 50 mM Tris HCl, pH 7.5, 80 mM KCl, 2 mM EDTA, 1 mMDTT, 40 μg/ml BSA and 0.01% Tween-20. Reactions were terminated by theaddition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mMEDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue].Samples were subjected to a 16% denaturing PAGE and dried gels wereexposed to a Phosphorlmager screen (GE Healthcare). Gel images werescanned using a Typhoon 8600 (GE Healthcare) and densitometric analyseswere performed using the ImageQuant software (GE Healthcare).

Surface Plasmon Resonance Analysis.

Binding experiments were performed as recently described. Briefly, Tdp1was amine coupled to a CM5 sensor chip (GE Healthcare, Piscataway N.J.).In order to protect the amine groups with the active site frommodification, 1 mM Tdp1 was incubated with 2 mM of a 14 baseoligonucleotide containing at phosphate group at the 3′ end(GATCTAAAAGACTT) (SEQ ID NO: 1) in 10 mM sodium acetate pH 4.5 for 20min. The CM5 chip surface was activated for 7 min with 0.1 M NHS and 0.4M EDC at a flow rate of 20 mL/min and Tdp1-oligonucleotide mixture wasinjected until approximately 4000 RU's was attached. Activated aminegroups were quenched with an injection of 1 M solution of ethanolaminepH 8.0 for 7 min. Any bound oligonucleotide was removed by washing thesurface with 1 M NaCl. A reference surface was prepared in the samemanner without coupling of Tdp1. Compound 70 was diluted into runningbuffer [10 mM Hepes, 150 mM NaCl, 0.01% tween 20 (v/v), 5% DMSO (v/v) pH7.5] and injected over all flow cells at 30 mL/min at 25° C. Followingcompound injections, the surface was regenerated with a 30 secondinjection 1 M NaCl, a 30 s injection of 50% DMSO (v/v) and a 30 srunning buffer injection. Each cycle of compound injection was followedby buffer cycle for referencing purposes. A DMSO calibration curve wasincluded to correct for refractive index mismatches between the runningbuffer and compound dilution series.

What is claimed is:
 1. A compound of the formula

or a pharmaceutically acceptable salt thereof, wherein: R^(T) is amino,R^(a) is 3-carboxymethylamino and R^(d) is hydrogen; or R^(T) is amino,R^(a) is 3-amino and R^(d) is 9-methoxy; or R^(T) is amino, R^(a) is3-iodo and R^(d) is hydrogen; or R^(T) is dimethylamino, R^(a) is 3-iodoand R^(d) is 9-methoxy; or R^(T) is dimethylamino, R^(a) is 3-cyano andR^(d) is hydrogen.
 2. A pharmaceutical composition comprising a compoundof claim 1 and one or more carriers, diluents, or excipients, or acombination thereof.
 3. A compound of the formula

or a pharmaceutically acceptable salt thereof, wherein: n is 1, 2, 3, 4,5, 6, 7, 8, 9, 11, or 12, R^(a) represents 1-4 substituents each ofwhich is independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted (1-4C)alkyl, optionallysubstituted (1-4C)alkoxy, cyano, nitro, optionally substitutedalkylthio, optionally substituted alkylsulfonyl, carboxylic acid andderivatives thereof, and sulfonic acid and derivatives thereof; or R^(a)represents 2-4 substituents where 2 of said substituents are adjacentsubstituents and are taken together with the attached carbons to form anoptionally substituted heterocycle, and where any remaining substituentsare each independently selected from the group consisting of hydrogen,halo, hydroxy, optionally substituted (1-4C)alkyl, optionallysubstituted (1-4C)alkoxy, cyano, nitro, optionally substituted(1-4C)alkylthio, optionally substituted (1-4C)alkylsulfonyl, carboxylicacid and derivatives thereof, and sulfonic acid and derivatives thereof;and R_(d) represents 1-4 substituents each of which is independentlyselected from the group consisting of hydrogen, halo, hydroxy,optionally substituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy,cyano, nitro, optionally substituted (1-4C)alkylthio, optionallysubstituted (1-4C)alkylsulfonyl, phenyl (which may bear one or moreamino, hydroxyl, halo, thiol, (1-6C)alkyl or halo(1-6C)alkylsubstituents), carboxylic acid and derivatives thereof, and sulfonicacid and derivatives thereof; or R_(d) represents 2-4 substituents where2 of said substituents are adjacent substituents and are taken togetherwith the attached carbons to form an optionally substituted heterocycle,and where any remaining substituents are each independently selectedfrom the group consisting of hydrogen, halo, hydroxy, optionallysubstituted (1-4C)alkyl, optionally substituted (1-4C)alkoxy, cyano,nitro, optionally substituted (1-4C)alkylthio, optionally substituted(1-4C)alkylsulfonyl, carboxylic acid and derivatives thereof, andsulfonic acid and derivatives thereof; R_(S) is (1-6C)alkyl,(3-7C)cycloalkyl or phenyl, which phenyl may bear one or more amino,hydroxyl, halo, thiol, (1-6C)alkyl or halo(1-6C)alkyl substituents.
 4. Apharmaceutical composition comprising a compound of claim 3 and one ormore carriers, diluents, or excipients, or a combination thereof.
 5. Acompound of the formula

or a pharmaceutically acceptable salt thereof, wherein: R^(T) isdimethylamino, R_(a) is 3-cyano and R_(d) is 8,9-methylenedioxy; orR^(T) is 4-morpholinyl, R_(a) is 3-cyano and R_(d) is hydrogen; or R^(T)is imidazolyl, R_(a) is 3-cyano and R_(d) is hydrogen.
 6. Apharmaceutical composition comprising a compound of claim 5 and one ormore carriers, diluents, or excipients, or a combination thereof.